NL2027815B1

NL2027815B1 - Genomic integration

Info

Publication number: NL2027815B1
Application number: NL2027815A
Authority: NL
Inventors: Catarina Martins Grandela Ana; Blanch Asensio Albert; Paul Frank Davis Richard
Original assignee: Academisch Ziekenhuis Leiden
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2022-10-07

Abstract

The present invention relates to a method of integrating an exogenous nucleic acid sequence into the genome of a cell, to a method of identifying a cell which has undergone a successful recombination event, and to a cell or a cell population produced by the method

Description

GENOMIC INTEGRATION

FIELD OF THE INVENTION The present invention relates to a method of integrating a desired nucleic acid sequence into the genome of a cell, particularly a mammalian cell, which provides for easier identification and more efficient selection of cells having successful integration of the nucleic acid sequence, and a reduction in unnecessary auxiliary sequences left in the genome after integration. The method is based on site -specific recombination techniques of integration, but novel positioning of the regulatory elements controlling the selectable marker provide advantages during selection, as does a further step of secondary recombination. The invention further relates to methods of selecting cells having successfully integrated nucleic acid sequences via the method, methods of producing a population of cells therefrom, a kit for performing the method, and a library of cells produced by the method. Component products such as vectors and cells used in the method are also provided.

INTRODUCTION There are a variety of editing tools available to perform integration of desired exogenous payload sequences into the genomic DNA of cells. However, many of the currently available methods have drawbacks.

Endonuclease-based gene editing systems, and in particular CRISPR-Cas9, have made it significantly easier to perform small-scale genomic modifications in cells (Ding et al. 2013). This includes the targeted knock- in of a fluorophore, correcting a point mutation, or disrupting a gene through short insertions or deletions ({indels) (Hockemeyer & Jaenisch 2016) . However, strategies for inserting multi-kilobase payloads that, for example comprise of several transgenes or a region of the genome, are limited with homology directed repair (HDR)-mediated integration efficiency significantly decreasing as the insert size increases (Shy et al. 2016; Gurumurthy & Perez-Pinera 2018).

In addition, when integrating such DNA fragments into genes not expressed in the mammalian cell, a selection marker is required to enrich for the targeted cells. However less than 10% of the cells following enrichment typically are targeted (Hockemeyer et al. 2011; Roberts et al. 2019). This is in part due to DNA fragment randomly integrating in the genome, thereby still expressing the selection marker for enrichment but at an off-target site.

Homology-independent integration strategies are capable of integrating larger DNA fragments but also have significant drawbacks. This includes inability to control directionality of integration, the occurrence of mutations at the insertion site which could influence expression of the integrated payload, as well as unintended integrations occurring at off-target sites (Brown et al. 2016) .

Retroviruses, lentiviruses and transposons are also commonly used for randomly inserting transgenes into the genome but there is no control over the insertion site, copy number or orientation of the transgene, which can subsequently lead to variegated expression in subpopulations of cells (Ellis 2005).

Similar issues also exist for generating panels of disease variants for functional annotation (van den Brink, Grandela et al. 2020). Such a platform would be enormously valuable for evaluating and appropriately classifying the pathogenicity of the numerous novel variants identified in patients for which their clinical relevance is currently unknown. However CRISPR-Cas9 based systems are too inefficient to perform such targeted genomic modifications on diploid genomic sequences at sufficient throughput, restricting such studies to haploid cell lines (Findlay et al. 2018).

Site-specific recombinases (SSRs) have proven useful tools for performing difficult genome engineering tasks in cultured mammalian cells such as the insertion, deletion or inversion of large DNA segments (Turan et al. 2013). SSRs achieve this by performing ‘cut and paste’ genome editing at precisely defined DNA sequences without requiring any additional co-factors, and are divided into two families based on the identity of the nucleophilic active site amino acid residue.

Phage-derived serine integrases, which includes the phiC31 and Bxbl recombinases, mediate unidirectional recombination between the enzyme’s unique phage and bacterial attachment site (attP and attB respectively) sequences, thereby enabling irreversible integration of transgenes into the genome (Brown et al. 2011). For example, it has been demonstrated that payloads of up to 27 kb can be inserted using Bxb1 recombinase in HEK 2937 cells (Duportet et al. 2014). Indeed, Bxb1 is the most effective integrase currently identified for catalysing recombination events in mammalian cells, with the additional advantage that Bxb1 target att sites are not naturally present in the human genome (Xu et al. 2013; Russell et al. 2006).

Tyrosine recombinases (e.g. Cre recombinase and flippases (Flp}} can also integrate DNA fragments {Anderson et al. 2012; Ordovás et al. 2015), although comparatively their integration efficiencies are lower and the event reversible (Turan et al. 2013}. However, both Cre and Flip are very efficient at excising cassettes flanked by directly repeated recombination sites, and so have found applications such as conditionally knocking out genes, removing selection cassettes following targeting, and in lineage tracing studies(Skarnes et al. 2011; Davis et al. 2008).

Despite both classes of SSRs being used to perform genetic modifications (Thyagarajan et al. 2008; Du et al. 2009; H. Zhu et al. 2013; F. Zhu et al. 2014; Ordovás et al. 2015; Farruggio et al. 2017; Chaudhari et al. 2020), a procedure for performing targeted integrations of large payloads (>10kb) at sufficient efficiency is still lacking. This is in part due to inefficient enrichment strategies, making positive selection of successful integrations difficult. Such SSR based methods typically rely on the use of a selectable marker linked to the payload, for example present in the integration vector with the payload, which maintains expression after a successful recombination to indicate the payload has integrated into the genome. Methods have evolved such that the selectable marker is only intended to be expressed after successful recombination. The most common way of achieving this is by separating the promoter and the sequence encoding the selectable marker. However, depending on the composition of the payload present (e.g. constitutively expressed transgenes), these methods can suffer from significant background expression from the sequence encoding the selectable marker, even when it is not combined with its promoter. This leads to problems in selecting those cells that have been successfully modified, and subsequent enrichment of homogenous cell populations that contain only successfully modified cells.

Furthermore, an SSR method that is suitable for multiplex assays involving DNA payloads >10 kb is still lacking, especially in mammalian stem cells. A cassette exchange strategy has been employed in most cases, whereby a previously targeted genomic fragment flanked by SSR recognition/attachment sites is exchanged for another similarly flanked transgene. While the exchange of fragments generally is quite efficient (Pei et al. 2015), the largest payload reported to be inserted is less than 10 kb (Zhu et al. 2014).

Alternatively, phiC31 and lambda integrases have been used to introduce plasmids into pseudo-attP sites present in the human genome (Liu et al. 2009; Farruggio et al. 2017; Chaudhari et al. 2020). Here the integration of larger inserts (up to ~20 kb} was achievable although it was not possible to target the plasmid to pre-selected genomic loci. Furthermore, locus-specific silencing of the transgenes occurred in some instances (Farruggio et al. 2017).

In addition, another major problem with such SSR methods is the continued presence of the vector backbone in the genome after recombination. Such excess genetic material can lead to post-integrative silencing (Tasic et al. 2011). This is essentially a by-product of performing genomic integrations which are highly undesirable in research, and especially undesired in any therapeutic applications.

The present invention is directed towards solving one or more of the above mentioned problems in the art. In particular, the present invention is directed towards providing an improved process for performing site specific recombination for integrating payloads, including those of large sizes, into the genome of cells, wherein the method provides accurate selection of those cells, and can be used to remove remaining backbone sequences from the genome.

STATEMENTS OF INVENTION According to a first aspect of the present invention, there is provided a method of integrating an exogenous nucleic acid sequence into the genome of a cell, the method comprising: (a) Providing a cell comprising a landing pad within its genome wherein the landing pad comprises in order from 5’ to 3’: a first recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon; (b} Introducing an integration vector into the cell, wherein the vector comprises (i) an exogenous nucleic acid sequence, and (ii) a construct comprising in order from 5’ to 3’: a promoter for driving expression of the selectable marker gene, a start codon, and a second recombination site; (c) Performing recombination between the first and second recombination sites According to a second aspect of the present invention, there is provided a method of identifying a cell which has undergone a successful recombination event, the method comprising: (a) Performing the method of the first aspect on one or more cells; (b) Identifying a cell which expresses the selectable marker gene According to a third aspect of the present invention, there is provided a method of making a population of cells which each comprise an exogenous nucleic acid sequence successfully integrated in the genome, the method comprising: (a) Performing the method of the first aspect on a plurality of cells; (b) identifying the cells that express the selectable marker; (c) Enriching the cells that express the selectable marker According to a fourth aspect of the present invention, there is provided a cell produced by the method ofthe first aspect. According to a fifth aspect, there is provided a cell comprising an exogenous nucleic acid sequence successfully integrated within its genome, the cell comprising a construct having, in order from 5 to 3": a promoter for driving expression of a selectable marker gene, a start codon, a recombination scar, and a nucleic acid sequence encoding the selectable marker gene.

According to a sixth aspect, there is provided a nucleic acid sequence encoding a landing pad comprising in order from 5’ to 3’: a recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon. According to a seventh aspect there is provided a landing pad vector comprising the nucleic acid 5 sequence of the sixth aspect. According to an eighth aspect there is provided a cell comprising a landing pad within its genome, wherein the landing pad comprises in order from 5’ to 3’: a recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon. According a ninth aspect, there is provided a nucleic acid sequence encoding an integration construct, the integration construct comprising in order from 5’ to 3’: a promoter for driving expression of a selectable marker gene, a start codon, and a recombination site, wherein the integration construct lacks a nucleic acid sequence encoding the selectable marker gene.

According to a tenth aspect there is provided an integration vector comprising (i) an exogenous nucleic acid sequence, and (ii) an integration construct according to the ninth aspect.

According to an eleventh aspect there is provided a cell comprising (i} a landing pad within its genome, wherein the landing pad comprises in order from 5’ to 3’: a first recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon; and (ii} an integration vector, wherein the vector comprises (i) an exogenous nucleic acid sequence, and (ii) a promoter for driving expression of the selectable marker gene, a start codon, and a second recombination site.

According to a twelfth aspect, there is provided a kit comprising a nucleic acid sequence encoding a landing pad of the sixth aspect, a landing pad vector of the seventh aspect or a cell of the eighth aspect; and a nucleic acid sequence encoding an integration construct of the ninth aspect or an integration vector of the tenth aspect; a recombinase enzyme or a nucleic acid sequence encoding a recombinase enzyme; and instructions for performing the method of the first aspect.

According to a thirteenth aspect, there is provided a library of cells according to the fifth aspect, or produced by the method of the first aspect or third aspect, wherein each cell comprises a different exogenous nucleic acid sequence.

Further features and embodiments of the aspects will now be described under the following sections. Any feature or embodiment in any section may be combined with any aspect in any workable combination.

DEFINITIONS The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

Such techniques are explained fully in the literature.

See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son inc, Library of Congress, USA}; Molecular Cloning: A Laboratory Manual, Third Edition, {Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.

J.

Gait ed., 1984}; U.S.

Pat.

No. 4,683,195; Nucleic Acid Hybridization {Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R.

Liss, Inc, 1987}; immobilized Cells and Enzymes (IRL Press, 1986}; Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology {Abelson and Simon, eds. -in-chief, Academic Press, Inc, New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols.

I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). To facilitate the understanding of this invention, a number of terms are defined below.

Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention.

Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration.

The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

The terms "comprising", “comprises” and "comprised of" as used herein are synonymous with "including", "includes" or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited features, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

As used herein, the term "promoter" refers to a region of DNA that generally is located upstream of a nucleic acid sequence to be transcribed that is needed for transcription to occur, i.e. which initiates transcription.

Promoters permit the proper activation or repression of transcription of a coding sequence under their control. A promoter typically contains specific sequences that are recognized and bound by plurality of TFs. TFs bind to the promoter sequences and result in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. A great many promoters are known in the art. A promoter will generally include a transcription start site having the sequence ‘ATG’, unless explicitly stated otherwise herein. In some embodiments of the invention a given promoter may lack a start codon, this is explicitly stated, and means that the promoter does not comprise a transcription start site. Suitably the promoter does not comprise an ‘ATG’ sequence. The term "nucleic acid" as used herein typically refers to an oligomer or polymer {preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C}, thymine (T) and uracil {U} which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose} groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid“ further preferably encompasses DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and/or partly or entirely, chemically or biochemically synthesised. A "nucleic acid" can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

“Transfection” in the present application refers broadly to any process of deliberately introducing nucleic acids into cells, and covers introduction of viral and non-viral vectors, and includes transformation, transduction and like terms and processes. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al.

(1986) Nature 319:791-3); lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; whiskers-mediated transformation; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e., that do not integrate into the genome of a host cell}, or can be vectors that integrate into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAC), yeast (YAC), or human (HAC)}) may be used to accommodate larger inserts. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

The term “operably linked”, “operably connected” or equivalent expressions as used herein refer to the arrangement of various nucleic acid elements relative to each such that the elements are functionally connected and are able to interact with each other in the manner intended. Such elements may include, without limitation, a promoter, an enhancer and/or a regulatory element, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed. The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of an expression product. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5’ terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements. As understood by the skilled person, operably linked implies functional activity, and is not necessarily related to a natural positional link. indeed, when used in nucleic acid expression cassettes, cis-regulatory elements will typically be located immediately upstream of the promoter (although this is generally the case, it should definitely not be interpreted as a limitation or exclusion of positions within the nucleic acid expression cassette), but this needs not be the case in vivo, e.g., a regulatory element sequence naturally occurring downstream of a gene whose transcription it affects is able to function in the same way when located upstream of the promoter. Hence, according to a specific embodiment, the regulatory or enhancing effect of the regulatory element is position- independent. A “spacer sequence” or “spacer” as used herein is a nucleic acid sequence that separates two functional nucleic acid sequences. It can have essentially any sequence, unless defined otherwise herein, provided it does not prevent the functional nucleic acid sequence from functioning as desired (e.g. this could happen if it includes a silencer sequence, prevents binding of the desired transcription factor, or suchlike). Typically, it is non-functional, as in it is present only to space adjacent functional nucleic acid sequences from one another, however in some cases the spacer may provide a minor non-coding function such as providing a cleavage sequence. Specific spacer sequences for use in the present invention are discussed elsewhere herein. “Cell culture”, as used herein, refers to a proliferating mass of cells that may be in either an undifferentiated or differentiated state “Complementary” or “complementarity”, as used herein, refers to the Watson-Crick base-pairing of two nucleic acid sequences. For example, for the sequence 5'-AGT-3' binds to the complementary sequence 3’-TCA-5’. Complementarity between two nucleic acid sequences may be “partial”, in which only some of the bases bind to their complement, or it may be complete as when every base in the sequence binds to its complementary base. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, the phrase “exogenous nucleic acid sequence” refers to a nucleic acid sequence originating from outside the organism or cell of the organism concerned. It may refer to a heterologous or homologous nucleic acid sequence. It may refer to a transgene. In one example, a transgene is a gene sequence, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable trait. In yet another example, the transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence.

The terms "subject” and "patient" are used interchangeably herein and refer to animals, preferably vertebrates, more preferably mammals, and specifically include human patients and non-human mammals. "Mammalian" subjects include, but are not limited to, humans. Preferred patients or subjects are human subjects.

A “therapeutic amount” or “therapeutically effective amount” as used herein refers to the amount of expression product effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect. The term thus refers to the quantity of an expression product that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Such amount will typically depend on the gene product and the severity of the disease, but can be decided by the skilled person, possibly through routine experimentation. As used herein, the terms “treat” or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. Beneficial or desired clinical results include, but are not limited to, prevention of an undesired clinical state or disorder, reducing the incidence of a disorder, alleviation of symptoms associated with a disorder, diminishment of extent of a disorder, stabilized (i.e., not worsening) state of adisorder, delay or slowing of progression of a disorder, amelioration or palliation of the state of a disorder, remission (whether partial or total}, whether detectable or undetectable, or combinations thereof. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. As used herein, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein he object is to bring a subject's body or an element thereof from an undesired physiological change or disorder to a desired state, such as a less severe or unpleasant state (e.g., amelioration or palliation), or back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical well-being of a subject), to keep it at said undesired physiological change or disorder (e.g., stabilization, or not worsening), or to prevent or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder.

As used herein the terms "prevention", "preventive treatment” or "prophylactic treatment" and the like encompass preventing the onset of a disease or disorder, including reducing the severity of a disease or disorder or symptoms associated therewith prior to affliction with said disease or disorder. Such prevention or reduction prior to affliction refers to administration of the nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein to a patient that is not at the time of administration afflicted with clear symptoms of the disease or disorder. "Preventing" also encompasses preventing the recurrence or relapse-prevention of a disease or disorder for instance after a period of improvement. In embodiments, the nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein may be for use in gene therapy.

DESCRIPTION Landing Pad Structure The method of the invention comprises a first step of providing a cell comprising a landing pad. The ‘landing pad’ is a term understood in the art to mean the location in a genome at which an exogenous nucleic acid sequence can be inserted, typically by recombination.

Suitably therefore the landing pad comprises a first recombination site. Suitably at which recombination may take place in the presence of a partner second recombination site and a recombinase enzyme. Suitably first and second recombination sites and corresponding recombinases are discussed hereinbelow.

Suitably the landing pad of the present invention further comprises a nucleic acid sequence encoding a selectable marker gene which lacks a start codon. Suitably the nucleic acid sequence encoding a selectable marker gene does not contain an ‘ATG’ codon prior to the open reading frame of the selectable marker gene. Suitably the landing pad also lacks a promoter for driving expression of the selectable marker gene. Advantageously, the lack of a start codon means that there is no possibility of any expression of the selectable marker gene prior to successful recombination. This in turn means that there is no background expression of the selectable marker. Therefore it is much easier and quicker to use the marker effectively to select cells having successful recombination events.

Suitably the nucleic acid sequence encoding a selectable marker gene which lacks a start codon is located downstream of the first recombination site. Suitably therefore, the landing pad comprises in order from 5’ to 3’: a first recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon.

Suitably the landing pad may further comprise a first spacer. Suitably the first spacer is located between the first recombination site and the nucleic acid sequence encoding a selectable marker gene lacking a start codon. Suitably therefore, the landing pad comprises in order from 5’ to 3’: a first recombination site, a first spacer, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon. Suitably the first spacer functions to ensure the start codon will be in-frame with the nucleic acid sequence encoding the selectable marker gene after recombination takes place.

Suitably the first spacer comprises a plurality of nucleotides. Suitably the first spacer comprises less than 20 nucleotides, suitably less than 15 nucleotides, suitably less than 10 nucleotides, suitably between 1-5 nucleotides, suitably 1, 2, 3, 4, or 5 nucleotides. Suitably the first spacer consists of 4 or 5 nucleotides.

Suitably the nucleotides may be any nucleotides.

However, suitably the first spacer does not comprise any start or stop codons in frame with the nucleic acid sequence encoding a selectable marker gene. Suitably, the first spacer does not comprise any start or stop codons. Suitably the first spacer does not comprise any of the following sequences: ATG, TAA, TGA, or TAG.

Suitably the first spacer may not be present, or suitably it may comprise one or more nucleotides. In one embodiment, the first spacer comprises 5 nucleotides. Suitably this may apply in an embodiment where the first recombination site is a BxB1 recombination site.

In one embodiment, the first spacer consists of the sequence: CATAT (SEQ ID NO: 1). Suitably the first spacer may comprise at least one nucleotide. Suitably the first spacer may further comprise additional nucleotides. Suitably the total number of additional nucleotides comprises a multiple of three. Suitably therefore, in such an embodiment, the first spacer comprises a sequence of N(Y)x, wherein N is a nucleotide and (Y) is an additional nucleotide, wherein xX’ represents the number of additional nucleotides and is a multiple of three. Suitably the total number of additional nucleotides may be 0,3, 6, 9, 12, 15, 18, 21 for example. In one embodiment, the total number of additional nucleotides is 3. In one such embodiment, the first spacer comprises a sequence of G(Y)s. Suitably, Y may consist of the sequence CGC. Suitably this may apply in an embodiment where the first recombination site is a phage C31 recombination site.

In one embodiment, the first spacer consists of the sequence GCGC (SEQ ID NO: 2). In one embodiment, the landing pad comprises in order from 5’ to 3’: a Bxb1 attP recombination site, a first spacer consisting of the sequence: CATAT, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon.

Suitably, the BxB1 attP recombination site and the first spacer may comprise the following sequence: GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACCCATAT (SEQ ID NO: 3) wherein the first spacer is indicated in bold.

In one embodiment, the landing pad comprises in order from 5’ to 3’: a phage C31 attP recombination site, a first spacer consisting of the sequence: GCGC, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon.

Suitably the phage C31 attP recombination site and the first spacer may comprise the following sequence: GTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTAGGCGC (SEQ ID NO: 4 ) wherein the first spacer is indicated in bold.

in some embodiments, the first spacer may comprise a sequence encoding a cleavage site, suitably a peptide cleavage site. In one embodiment, the first spacer may comprise a 2A peptide cleavage sequence. Suitably the landing pad may further comprise a third recombination site. Suitably the third recombination site is upstream of the first recombination site. Suitably the third recombination site may be a tyrosine recombinase site such as loxP, loxP257, FRT, FRT-F3, rox as explained below. Other derivative tyrosine recombinase sites may also be used, for example derivative lox or FRT or rox sites. Suitably the landing pad may comprise a fourth recombination site. Suitably the fourth recombination site is downstream of the nucleic acid sequence encoding a selectable marker gene which lacks a start codon. Suitably the fourth recombination site may be a tyrosine recombinase site such as loxP, loxP257, FRT, FRT-F3 or rox as explained below.

in one embodiment, therefore, the landing pad comprises in order from 5’ to 3’: a third recombination site, a first recombination site, a first spacer, a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, and a fourth recombination site.

Suitably the third and fourth recombination sites are for removal of auxiliary sequences after integration of the integration vector into the genome of the cell.

Suitably the landing pad may be located in any suitable position in the genome of the cell. Suitably the landing pad is located at a position in the genome of the cell where it is desired to insert the exogenous nucleic acid sequence.

Suitably the landing pad is located at a position where it does not disrupt the coding sequence of any other gene, suitably in a non-coding region.

A suitable landing pad location may be in an intron, or non-coding region of the genome. Suitably that does not contain any regulatory sequences or epigenetic modifications. Such landing pad locations may be termed ‘safe harbours’. Suitable safe harbours may be known in the genome. Suitable safe harbours are generally defined as having one or more of the following features:

1.>300 kb from any cancer-related gene, for example those on the allOncogenes list

2. >300 kb from any miRNA/other functional small RNAs

3. >50 kb from any 5’ gene end 4, >50 kb away from any replication origin

5. >50 kb away from any ultra-conserved element

6. Low transcriptional activity (no mRNA —25 kb)

7. Not in a copy number variable region

8. In open chromatin (DHS signal -1 kb)

9. Unique {one copy in human genome) Suitably the landing pad location may comprise all of these features. In one embodiment, the landing pad is located in intron 1 of PPPIR12C. In one embodiment, the landing pad location is the ‘AAVS1’ location. Suitably the method may further comprise a step of producing a cell comprising the landing pad within its genome. Suitably such a step may occur prior to step (a) of the method. Suitably this step may comprise inserting the landing pad into the genome of a cell. Suitably such insertion may be achieved by any suitable means. Suitably such insertion may be achieved by recombination. Alternatively, such an insertion may be achieved by CRISPR-Cas9 methods. Suitably the landing pad may be inserted into the genome by cleaving the genomic DNA at the desired location and inserting the landing pad. Suitably the DNA may be cleaved by using an endonuclease enzyme, suitably a Cas endonuclease. Suitably a Cas9 endonuclease may be used. Suitably a CRISPR-Cas9 system may be used. Suitably the Cas endonuclease is accompanied by a DNA-targeting RNA, suitably a guide RNA. Suitably the guide RNA comprises a sequence which is complementary to the location in the genomic DNA to be cleaved by the Cas endonuclease, the ‘cleavage site’, suitably to the location in the genomic DNA at which the landing pad is to be inserted. Suitably a Cas9-gRNA complex is used to cleave the genomic DNA at the desired location, i.e. at the cleavage site.

In one embodiment, the guide RNA and the Cas protein form a complex that specifically targets DNA.

In one embodiment, the DNA binding protein is a Cas protein.

Exemplary Cas proteins include Cpft, C2c1, C2c3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Cas13b, and Casl3c.

Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas100, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpfl, C2c1, C2C3, Casl2a, Cas12b, Casl2c, Cas12d, Casl2e, Cas13a, Cas13b, and Cas13c. in one embodiment of any aspect, the Cas protein is Cas9. For example, the Cas9 protein may be a type |, Il, or lil Cas9 protein, suitably a type II Cas9 protein.

The Cas9 protein may be a modified Cas9 protein or a wild type Cas9 protein.

An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein.

In one embodiment of any aspect, the Cas protein is a Cas9 variant selected from Staphylococcus aureus (SaCas9), Streptococcus thermophilus (StCas9), Neisseria meningitidis {(NmCas9), Francisella novicida {FnCas9), and Campylobacter jejuni (CjCas9). in one embodiment, the guide RNA may be a single guide RNA.

The sgRNA is configured to form a stable complex with Cas9. The guide RNA comprises a DNA binding region and a protein binding region.

The protein binding region binds to the Cas9 protein.

The DNA binding region is complementary to the target DNA, and suitably comprises at least 15 nucleotides, suitably between 15 and 20 nucleotides.

In one embodiment, the guide RNA comprises a DNA binding region of about 20 nucleotides.

The DNA binding region of the guide RNA is complementary to the target DNA sequence.

In one embodiment, the DNA binding region of the guide RNA is complementary to the non-template strand of the target DNA.

Generally, the DNA binding region of the guide RNA is designed to complement or substantially complement the target DNA.

Suitably the DNA binding region of the guide RNA may be designed to target a particular DNA sequence as long as the target DNA sequence comprises a PAM.

In one embodiment, the PAM sequence is NGG, suitably when the Cas9 protein is from Streptococcus pyogenes . The use of such a dead Cas9 and guide RNA to target and bind DNA is understood as CRISPR interference (Qi, L.

S.; Larson, M.

H.; Gilbert, L.

A,; Doudna, J.

A.; Weissman, J.

S.; Arkin, A.

P.; Lim, W.

A.

Cell. 152 (5): 1173-1183. 2013)). Suitable guide RNAs may be designed to target Cas proteins to bind to a particular sequence by the use of known software.

An exemplary gRNA sequence for the gRNA landing pad in intron 1 of PPPIR12C is: 5'- GGGGCCACTAGGGACAGGAT (SEQ ID NO: 5).

Suitably therefore the step of producing a cell comprising the landing pad within its genome may comprise introducing into the cell a nucleic acid sequence encoding the landing pad, a Cas9 endonuclease, and a gRNA. Suitably the nucleic acid sequence encoding the landing pad, a Cas9 endonuclease, and a gRNA may be introduced into the cell in any format, for example as a nucleic acid sequence upon a vector, an mRNA, or as a protein complex.

In some embodiments, the nucleic acid sequence encoding the landing pad is introduced on a vector, suitably a landing pad vector according to the invention. In some embodiments, the Cas9-gRNA is introduced as a protein complex. Suitably wherein the gRNA comprises a sequence complementary to the desired landing pad location in the genome.

Suitably, in such embodiments, the nucleic acid sequence encoding the landing pad further comprises arm regions located at the 5" and 3’ ends of the nucleic acid sequence. Suitably the arm regions have high homology to the genomic DNA of the landing pad. Suitably the arm regions have high homology to the genomic DNA on either side of the cleavage site, suitably high homology to the 5’ and 3’ flanks surrounding the cleavage site at the landing pad location. Suitably the arm regions are up to 1000bp in length, suitably up to 500bp in length, suitably up to 250bp in length, suitably up to 100bp in length, suitably up to 50bp in length, suitably up to 25bp in length, suitably up to 10bp in length. in one embodiment, therefore, the landing pad comprises in order from 5’ to 3’: a 5’ arm region, a third recombination site, a first recombination site, a first spacer, a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, a fourth recombination site, and a 3’ arm region.

Suitably the landing pad may further comprise an additional selectable marker gene. Suitably under the control of a promoter. Suitably such an additional selectable marker gene allows for selection of cells that have successfully integrated the landing pad into the genome. Suitably after producing a cell comprising the landing pad within its genome, cells expressing the additional selectable marker are selected as described elsewhere herein, for use in the recombination step of the method.

Suitably the additional selectable marker gene is present upstream of the first recombination site. Suitably the additional selectable marker gene is present downstream of the third recombination site.

Suitable selectable marker genes are discussed elsewhere herein. In one embodiment, the additional selectable marker gene is a fluorescent reporter gene. In one embodiment, therefore, the landing pad comprises in order from 5’ to 3’: a third recombination site, an additional selectable marker gene under the control of a promoter, a first recombination site, a first spacer, a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, and a fourth recombination site. Suitably the landing pad vector of the invention may comprise any of the landing pad sequences as described herein. Integration Vector Structure The method of the invention comprises a second step of introducing an integration vector into the cell comprising the landing pad. Suitably the integration vector is used to transport the exogenous nucleic acid sequence into the cell so that it can subsequently be integrated into the genome of the cell. Suitably therefore the integration vector comprises an exogenous nucleic acid sequence. Exogenous nucleic acids that it may be desirable to integrate into the genome are described elsewhere herein. In addition to the exogenous nucleic acid sequence, suitably the integration vector further comprises a construct comprising a promoter for driving expression of the selectable marker gene, a start codon, and a second recombination site.

Suitably the promoter is for driving expression of the selectable marker gene present in the landing pad. Suitable such promoters are described elsewhere herein. Suitably the integration vector does not itself comprise a nucleic acid sequence encoding a selectable marker. Suitably the start codon is downstream of the promoter. Suitably upon successful recombination, the promoter and start codon are placed upstream and in-frame with the nucleic acid sequence encoding a selectable marker gene which lacks a start codon. Suitably they are operably linked. Suitably then expression of the selectable marker gene is possible. Advantageously, this means that expression of the nucleic acid sequence encoding a selectable marker gene only occurs after a successful recombination event.

Suitably the second recombination site is downstream of the start codon. Suitably at which recombination may take place in the presence of a partner first recombination site and a recombinase enzyme. Suitably first and second recombination sites and corresponding recombinases are discussed hereinbelow.

Suitably therefore, the integration vector comprises a construct comprising in order from 5’ to 3’: a promoter for driving expression of the selectable marker gene, a start codon, and a second recombination site.

Suitably the integration vector may further comprise a second spacer. Suitably the second spacer is located between the start codon and the second recombination site. Suitably therefore, the integration vector comprises a construct having in order from 5’ to 3’: a promoter for driving expression of the selectable marker gene, a start codon, a second spacer, and a second recombination site. Suitably the second spacer functions to ensure the start codon will be in-frame with the nucleic acid sequence encoding the selectable marker gene after recombination takes place.

Suitably both the first and second spacers function together to ensure that the start codon will be in- frame with the nucleic acid sequence encoding the selectable marker gene after recombination takes place.

Suitably the second spacer comprises a plurality of nucleotides. Suitably the second spacer comprises less than 20 nucleotides, suitably less than 15 nucleotides, suitably less than 10 nucleotides, suitably between 1-5 nucleotides, suitably 1, 2, 3, 4, or 5 nucleotides. Suitably the second spacer consists of 1 or 2 nucleotides. Suitably the nucleotides may be any nucleotides.

However, suitably the second spacer does not comprise any start or stop codons in frame with the nucleic acid sequence encoding a selectable marker gene. Suitably, the second spacer does not comprise any start or stop codons. Suitably the second spacer does not comprise any of the following sequences: ATG, TAA, TGA, or TAG.

Suitably the second spacer may not be present, or may comprise one or more nucleotides. In one embodiment, the second spacer comprises one nucleotide. Suitably in such an embodiment, the total number of nucleotides comprised in the first and the second spacer is a multiple of three. Suitably the total number of nucleotides in the first spacer and the second spacer may be 0, 3,6, 9, 12, 15, 18, 21 for example. in one embodiment, the total number of nucleotides in the first spacer and the second spacer is 6. Suitably the total number of nucleotides may be distributed between the first and second spacer in any combination. For example, the first spacer may comprise 4 nucleotides, and the second spacer may comprise 2 nucleotides or vice versa, alternatively the first spacer and second spacer may each comprise 3 nucleotides. In one embodiment, the first spacer consists of 5 nucleotides, and the second spacer consists of 1 nucleotide. Suitably this may apply in an embodiment where the second recombination site is a BxB1 recombination site.

In one embodiment, the second spacer consists of the sequence G.

Suitably the second spacer comprises at least two nucleotides. Suitably the second spacer may further comprise additional nucleotides. Suitably the total number of additional nucleotides comprises a multiple of three. Suitably therefore, in such an embodiment, the second spacer comprises a sequence of (YIxNN(Y)x, wherein N is a nucleotide and (Y) is an additional nucleotide, wherein ‘x’ represents the number of additional nucleotides and is a multiple of three. Suitably the total number of additional nucleotides may be 0, 3, 6, 9, 12, 15, 18, 21 for example. In one embodiment, the total number of additional nucleotides is 0. In one such embodiment, the second spacer comprises a sequence of {Y)oGC(Y)o. Suitably this may apply in an embodiment where the second recombination site is a phage C31 recombination site.

In one embodiment, the second spacer consists of the sequence GC.

in one embodiment, the integration vector comprises a construct comprising in order from 5’ to 3’: a promoter for driving expression of the selectable marker gene, a start codon, a second spacer consisting of G, and an BxB1 attB recombination site.

Suitably, the start codon, second spacer and attB recombination site comprise the following sequence: ATGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCC (SEQ ID NO: 6) wherein the start codon is underlined and the second spacer is in bold.

In one embodiment, the integration vector comprises a construct comprising in order from 5’ to 3’: a promoter for driving expression of the selectable marker gene, a start codon, a second spacer consisting of GC, and a phage C31 attB recombination site.

Suitably, the start codon, second spacer and phage C31 attB recombination site comprise the following sequence:

ATGGCCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCACCTCACCCATC {SEQ ID NO: 7) wherein the start codon is underlined and the second spacer is in bold.

In some embodiments, the second spacer may comprise a sequence encoding a cleavage site, suitably a peptide cleavage site. in one embodiment, the second spacer may comprise a 2A peptide cleavage sequence.

Suitably the integration vector may further comprise a fifth recombination site. Suitably the fifth recombination site is upstream of the promoter for driving expression of the selectable marker gene.

Suitably the fifth recombination site may be a tyrosine recombinase site such as loxP, loxP257, FRT, FRT-

F3, or rox as explained below. Other derivative tyrosine recombinase sites may also be used, for example derivative lox or FRT or rox sites. Suitably the integration vector may further comprise a sixth recombination site. Suitably the sixth recombination site is downstream of the second recombination site. Suitably the sixth recombination site may be a tyrosine recombinase site such as loxP, loxP257, FRT, FRT-F3, or rox as explained below. Other derivative tyrosine recombinase sites may also be used, for example derivative lox or FRT or rox sites. Suitably the fifth and sixth recombination sites are part of the construct of the integration vector, or the nucleic acid sequence encoding the integration construct. in one embodiment, therefore, the integration vector or the nucleic acid sequence encoding the integration construct comprises a construct having in order from 5’ to 3’: a fifth recombination site, a promoter for driving expression of the selectable marker gene, a start codon, a second spacer, a second recombination site, and a sixth recombination site. Suitably, in addition, the integration vector comprises an exogenous nucleic acid sequence {payload). First Recombination The method of the invention comprises a step of performing recombination between the first and second recombination sites. Suitably this step integrates the vector into the genome of the cell. Suitably the vector is inserted into the genome of the cell. Suitably at the position of the first recombination site in the landing pad. Suitably this step may be regarded as the first recombination step of the method.

Suitably the first recombination step is performed by a first recombinase enzyme. Suitably any recombinase enzyme may be used as the first recombinase. Suitably the first recombinase is a serine recombinase. Suitable serine recombinases include: BxB1 or PhiC31, R4, phiBT1, WB, SPBc, TP901, and TG1, for example. In other embodiments, a tyrosine integrase may be used as the first recombinase. Suitable tyrosine integrases may include Hk022 or Lambda phage integrase, for example.

In one embodiment, the first recombination step is performed by a BxB1 recombinase. In one embodiment, the first recombination step is performed by a PhiC31 recombinase. Suitably the first and second recombination sites are recombination sites recognised by the first recombinase enzyme. Suitably the first and second recombination sites are interchangeable.

Suitably the first and second recombination sites are selected from either attP or attB.

In one embodiment, the first recombination site is attP.

In one embodiment, the second recombination site is attB.

In one embodiment, the first recombination site is BxB1 attP, and the second recombination site is BxB1 attB. in one embodiment, the first recombination site is PhiC31 attP, and the second recombination site is PhiC31 attB.

Suitably the first recombinase is introduced into the cell.

Suitably therefore the method comprises a step of introducing the first recombinase into the cell, suitably prior to the first recombination step.

Suitably the first recombinase may be introduced into the cell as a nucleic acid sequence encoding the first recombinase or alternatively as a protein.

In embodiments where a nucleic acid sequence encoding the first recombinase is introduced into the cell, suitably it may be comprised on a vector.

Suitable methods and vectors are described hereinbelow.

Alternatively it may be introduced as mRNA, using any of the transfection methods described below.

Suitably, the mRNA may be encapsulated, for example in a liposome or otherwise.

In embodiments where the recombinase is introduced into the cell as a protein, suitably the protein is introduced by any of the transfection methods described below. in one embodiment, the method may comprise: (a) Providing a cell comprising a landing pad within its genome wherein the landing pad comprises in order from 5’ to 3’: a first recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon; (b} Introducing an integration vector into the cell, wherein the vector comprises (i) an exogenous nucleic acid sequence, and (ii) a construct comprising in order from 5’ to 3’: a promoter for driving expression of the selectable marker gene, a start codon, and a second recombination site; (c) Introducing a first recombinase into the cell; (d} Performing recombination between the first and second recombination sites using the first recombinase.

Second Recombination The method of the invention may further comprise a second step of performing recombination.

Suitably after the first recombination step.

Suitably the second recombination step comprises performing recombination between additional recombination sites located in the landing pad and the integration vector.

Suitably the landing pad may comprise a third recombination site, and a fourth recombination site, as discussed hereinabove. Suitably the integration vector may comprise a fifth recombination site and a sixth recombination site as discussed hereinabove.

Suitably the second recombination step comprises performing recombination between the third and sixth recombination sites, and between the fourth and fifth recombination sites. Suitably recombination between the third and sixth recombination sites, and between the fourth and fifth recombination sites may take place simultaneously or sequentially. In one embodiment, recombination between the third and sixth recombinant sites may take place before recombination between the fourth and fifth recombination sites. Alternatively, recombination between the fourth and fifth recombination sites may take place before recombination between the third and sixth recombinant sites.

In one embodiment, the method of the invention may comprise: (a) Providing a cell comprising a landing pad within its genome wherein the landing pad comprises in order from 5’ to 3’: (a third recombination site), a first recombination site, and a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, (a fourth recombination site); (b} Introducing a vector into the cell, wherein the vector comprises (i) an exogenous nucleic acid sequence, and (ii} a construct comprising in order from 5’ to 3’: (a fifth recombination site), a promoter for driving expression of the selectable marker gene, a start codon, and a second recombination site, (a sixth recombination site}; (c) Performing recombination between the first and second recombination sites; (d) Performing recombination between the third and sixth recombination sites, and between the fourth and fifth recombination sites.

Suitably the second recombination step removes auxiliary nucleic acid sequences from the genome of the cell. Auxiliary nucleic acid sequences may include parts of the landing pad, or parts of the integration vector, suitably the auxiliary nucleic acid sequences that are no longer required after integration of the exogenous nucleic acid sequence. Suitable auxiliary nucleic acid sequences it is desirable to remove may include: the nucleic acid sequence encoding the selectable marker, the nucleic acid sequence encoding any additional selectable markers, promoters driving expression of any selectable markers, recombination scars such as attL: or attR, first and second spacers, origin of replication and the like.

Suitably, after the second recombination step, less than 500bp of auxiliary nucleic acid sequences remain in the genome, suitably less than 400bp, suitably less than 300bp, suitably less than 200bp, suitably less than 100bp.

Suitably the second recombination step is performed by a second and optionally a third recombinase enzyme, Suitably any recombinase enzyme may be used as a second or third recombinase. Suitably the second and third recombinases are tyrosine recombinases. Suitable tyrosine recombinases include Cre, Flp, Dre, KD, B3, Kw, VCre, SCre, or Vika recombinase, for example. Suitably, the second and third recombinase may be the same recombinase. Optionally, however, the second and third recombinase may be different recombinases.

Suitably the third and sixth recombination sites are recombination sites recognised by a second recombinase enzyme. Suitably the third and sixth recombination sites are interchangeable. Suitably the fourth and fifth recombination sites are recombination sites recognised by a third recombinase enzyme. Suitably the fourth and fifth recombination sites are interchangeable.

in one embodiment, the method of the invention may comprise: (a) Providing a cell comprising a landing pad within its genome wherein the landing pad comprises in order from 5’ to 3’: a third recombination site, a first recombination site, a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, a fourth recombination site; (b} Introducing a vector into the cell, wherein the vector comprises (i} an exogenous nucleic acid sequence, and (ij) a construct comprising in order from 5’ to 3’: a fifth recombination site, a promoter for driving expression of the selectable marker gene, a start codon, a second recombination site, and a sixth recombination site; (c} Performing recombination between the first and second recombination sites with a first recombinase enzyme; (d} Performing recombination between the third and sixth recombination sites with a second recombinase enzyme, and between the fourth and fifth recombination sites with the second recombinase enzyme or a third recombinase enzyme.

Suitably the third and sixth recombination sites may be loxP, loxP257, FRT, FRT-F3, or rox, or variants thereof.

Suitably the fourth and fifth recombination sites may be loxP, loxP257, FRT, FRT-F3, or rox, or variants thereof.

However, suitably the third and sixth recombination sites are the same, and the fourth and fifth recombination sites are the same. Suitably the third and sixth recombination sites are different to the fourth and fifth recombination sites.

in one embodiment, the third and sixth recombination sites are loxP. Suitably in such an embodiment, the second recombinase is a Cre recombinase. In another embodiment, the third and sixth recombination sites are FRT. Suitably in such an embodiment, the second recombinase is a Flp recombinase.

in one embodiment, the fourth and fifth recombination sites are loxP257. Suitably in such an embodiment, the third recombinase is also a Cre recombinase. In another embodiment, the fourth and fifth recombination sites are FRT-F3. Suitably in such an embodiment, the third recombinase is also a Flp recombinase.

Suitably in an embodiment where the third and sixth recombination sites are loxP and the fourth and fifth recombination sites are loxP257, only a second recombinase is required, and suitably the second recombinase is a Cre recombinase.

Suitably in an embodiment where the third and sixth recombination sites are FRT, and the fourth and fifth recombination sites are FRT-F3, only a second recombinase is required, and suitably the second recombinase is a Flp recombinase.

Suitably, therefore, in some embodiments the second and third recombinase may be the same. Suitably therefore only a second recombinase needs to be provided to the cell. This may occur when all of the third to sixth recombination sites are recognised by the same recombinase enzyme.

Alternately, in an embodiment where the third and sixth recombination sites are loxP or loxP257 and the fourth and fifth recombination sites are FRT or FRT-F3, a second recombinase and a third recombinase are required, suitably the second recombinase is a Cre recombinase and the third recombinase is a Fip recombinase.

Equally, in an alternate embodiment where the third and sixth recombination sites are FRT or FRT-F3 and the fourth and fifth recombination sites are loxP or loxP257, a second recombinase and a third recombinase are required, suitably the second recombinase is a Flp recombinase and the third recombinase is a Cre recombinase.

Suitably the second and optional third recombinases are introduced into the cell. Suitably therefore the method may comprise a step of introducing the second recombinase into the cell, and a step of introducing the third recombinase into the cell. In embodiments where the second and third recombinases are the same, the method may only comprise a step of introducing a second recombinase into the cell. Suitably after the first recombination step of the method. Suitably the second and optional third recombinase may be introduced into the cell in the same step or subsequent steps. Suitably the second and optional third recombinases may be introduced into the cell as one or more nucleic acid sequences encoding the second and optional third recombinase, or alternatively as proteins. In embodiments where nucleic acid sequences encoding the second and optional third recombinases are introduced into the cell, suitably they may be comprised on one or more vectors. Suitably one vector may comprise the nucleic acid sequences encoding the second and optionally the third recombinase, or multiple vectors may comprise the nucleic acid sequences encoding the second and third recombinases. Suitable methods and vectors are described hereinbelow. Alternatively the nucleic acid sequence encoding the second and optionally the third recombinase may be introduced as mRNA, using any of the transfection methods described below. Suitably, the mRNA may be encapsulated, for example in a liposome or otherwise. In embodiments where the second and optional third recombinase are introduced into the cell as proteins, suitably the proteins are introduced by any of the transfection methods described below.

In one embodiment, the method of the invention may comprise: (a) Providing a cell comprising a landing pad within its genome wherein the landing pad comprises in order from 5’ to 3’: a third recombination site, a first recombination site, a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, and a fourth recombination site; {b} Introducing a vector into the cell, wherein the vector comprises (i) an exogenous nucleic acid sequence, and (ii} a construct comprising in order from 5 to 3’: a fifth recombination site, a promoter for driving expression of the selectable marker gene, a start codon, a second recombination site, and a sixth recombination site; {c) Introducing a first recombinase into the cell; (d} Performing recombination between the first and second recombination sites using the first recombinase enzyme; (e} Introducing a second recombinase into the cell;

(f) Performing recombination between the third and sixth recombination sites using the second recombinase enzyme, and between the fourth and sixth recombination sites using the second recombinase enzyme.

in embodiments where the second and third recombinases are not the same, the method of the invention may comprise: (a) Providing a cell comprising a landing pad within its genome wherein the landing pad comprises in order from 5’ to 3’: a third recombination site, a first recombination site, a nucleic acid sequence encoding a selectable marker gene which lacks a start codon, and a fourth recombination site; {(b} Introducing a vector into the cell, wherein the vector comprises (i) an exogenous nucleic acid sequence, and (ii) a construct comprising in order from 5’ to 3’: a fifth recombination site, a promoter for driving expression of the selectable marker gene, a start codon, a second recombination site, and a sixth recombination site; {c} Introducing a first recombinase into the cell; (d} Performing recombination between the first and second recombination sites using the first recombinase enzyme; (e} Introducing a second recombinase and a third recombinase into the cell; (f) Performing recombination between the third and sixth recombination sites using the second recombinase enzyme, and between the fourth and sixth recombination sites using the third recombinase enzyme.

Suitably the second step of recombination produces a cell comprising an exogenous nucleic acid sequence successfully integrated within its genome, and minimal auxiliary sequences remain. Such cells are described hereinbelow.

Suitably the kit of the invention may therefore comprise more than one recombinase or nucleic acid sequence encoding a recombinase. Suitably if the second step of recombination is to be carried out the kit comprises at least two recombinase enzymes or nucleic acid sequences encoding at least two recombinase enzymes. Suitably a first and a second recombinase, optionally a third recombinase, as described herein.

Resulting Construct The method of the invention is intended to produce a cell which comprises an exogenous nucleic acid sequence successfully integrated within its genome. Suitably the cell produced by the method of the first aspect, in addition to the exogenous nucleic acid sequence, further comprises a construct having, in order from 5’ to 3’: a promoter for driving expression of a selectable marker gene, a start codon, a recombination scar, and a nucleic acid sequence encoding the selectable marker gene. Suitably such a cell is in accordance with the fifth aspect of the invention.

Suitably the promoter and start codon are in frame with the nucleic acid sequence encoding the selectable marker gene. Suitably the promoter and the start codon are now operably linked to the selectable marker gene. Suitably therefore the selectable marker gene is expressed from the construct.

Suitably the recombination scar is a nucleic acid sequence resulting from the first recombination step of the method.

Suitably the recombination scar comprises a first part formed from the second recombination site and a second part formed from the first recombination site. Suitably the first part comprises a part of the nucleic acid sequence of the second recombination site. Suitably the second part comprises a part of the nucleic acid sequence of the first recombination site. Suitably the first part comprises about half of the nucleic acid sequence of the second recombination site. Suitably the second part comprises about half of the nucleic acid sequence of the first recombination site.

Suitably the recombination scar is an attR or attL site.

Suitably the recombination scar comprises a first part formed from the attB recombination site and a second part formed from the attP recombination site. Suitably therefore the recombination scar is an attL site.

In one embodiment the recombination scar is an attL site. In one embodiment, the recombination scar is formed from BxB1 attB and BxB1 attP recombination sites, therefore the recombination scar is a BxB1 attL site. In one embodiment, the recombination scar is formed from phiC31 attB and phiC31 attP recombination sites, therefore the recombination scar is a phiC31 attL site.

Surprisingly, the inventors have found that the recombination scar is well tolerated in the resulting construct and the selectable marker gene is expressed despite the recombination scar being located between the start codon and the nucleic acid sequence encoding the gene. Such integration and landing pad constructs in which the start codon of the selectable marker gene has been separated from the coding sequence of the selectable marker gene have not previously been attempted as a means to control expression of a selectable marker gene during genomic integration processes. The inventors have found that as long as suitable spacers are present to ensure the start codon is in frame with the nucleic acid sequence encoding the selectable marker gene after recombination, that this is an effective means of control. Suitably therefore, the cell construct may further comprise a first and a second spacer as defined hereinabove.

Suitably the cell comprises a construct having, in order from 5’ to 3’: a promoter for driving expression of a selectable marker gene, a start codon, a second spacer, a recombination scar, a first spacer, and a nucleic acid sequence encoding the selectable marker gene.

In one embodiment, the cell comprises a construct having, in order from 5’ to 3’: a promoter for driving expression of a selectable marker gene, a start codon, a second spacer, an attL recombination scar, a first spacer, and a nucleic acid sequence encoding the selectable marker gene. In one embodiment, the cell comprises a construct having, in order from 5’ to 3’: a promoter for driving expression of a selectable marker gene, a start codon, a second spacer consisting of the sequence G, a BxB1 attL recombination scar, a first spacer consisting of the sequence CATAT, and a nucleic acid sequence encoding the selectable marker gene. Suitably the start codon, second spacer, attL recombination scar, and first spacer may comprise the following sequence: ATGGCCGGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACCCATAT (SEQ ID NO: 8) wherein the start codon is underlined and the second and first spacers are in bold. in one embodiment, the cell comprises a construct having, in order from 5’ to 3’: a promoter for driving expression of a selectable marker gene, a start codon, a second spacer consisting of the sequence GC, a PhiC31 attL recombination scar, a first spacer consisting of the sequence GCGC, and a nucleic acid sequence encoding the selectable marker gene. Suitably the start codon, second spacer, attL recombination scar, and first spacer may comprise the following sequence: ATGGCCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGAGTTCTCTCAGTTGGGGGCGTAGGCGC (SEQ ID NO: 9) wherein the start codon is underlined and the second and first spacers are in bold. Suitably the cell construct may further comprise a second recombination scar, the third, fourth, fifth, and sixth recombination sites if present, and any additional selectable marker present in the landing pad. In one embodiment, the cell comprises in order from 5’ to 3’: the third recombination site, an attR recombination scar, a sixth recombination site, the exogenous nucleic acid sequence, a fifth recombination site, a promoter for driving expression of a selectable marker gene, a start codon, an attL recombination scar, a nucleic acid sequence encoding the selectable marker gene, and a fourth recombination site.

Optionally any additional selectable marker present in the landing pad may be comprised between the third recombination site and the attR recombination scar.

Suitably the attR and attL sites may be switched.

Suitably the cell produced after the second recombination step which may take place after the method of the first aspect as described herein comprises fewer auxiliary sequences.

Suitably the cell produced after the second recombination step comprises an exogenous nucleic acid sequence successfully integrated within its genome, and auxiliary sequences.

Suitably less than 300bp of auxiliary sequences as defined elsewhere herein.

Suitably such a cell comprises in order from 5’ to 3’: a third recombination site, an exogenous nucleic acid sequence, and a fourth recombination site.

Exogenous Nucleic Acid Sequence The method of the invention effectively integrates exogenous nucleic acid sequences into the genome of cells.

In some cases the exogenous nucleic acid sequence may be regarded as a ‘payload’. In some cases, for example in the aspects referring to an integration vector alone, the exogenous nucleic acid may simply be regarded as a nucleic acid sequence of interest, sutiably for insertion into the genome of a cell.

Suitably the exogenous nucleic acid sequence may be any nucleic acid sequence, suitably any nucleic acid sequence that it is desirable to integrate into the genome of a cell.

Suitably the exogenous nucleic acid sequence may be of any size.

Suitably the exogenous nucleic acid sequence may have no upper limit in size.

Suitably the exogenous nucleic acid sequence may be from 1 or 2kb up to about 250kb in size.

Suitably 1kb, 2kb, 5kb, 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 150kb, 200kb, 250kb, or integer value therebetween.

Suitably the exogenous nucleic acid sequence may be from about 27kb to 173kb in size.

Advantageously, the inventors have found the present method can be used to integrate much larger nucleic acid sequences than previously expected.

Previous methods based on site specific recombination have integrated a maximum size of around 27kb into the genome of a cell, however the inventors have shown that 173kb can be integrated successfully.

Suitably the exogenous nucleic acid sequence may comprise one or more genes. Suitably one or more genes which encode one or more expression products, for example one or more proteins which it is desired to express in the cell.

Suitably the protein may be an advantageous protein, for example a therapeutic protein. Suitably therefore the exogenous nucleic acid sequence may encode a therapeutic protein. Suitable therapeutic proteins are: antibodies or derivatives/parts thereof, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, thrombolytics and the like.

in one embodiment, the exogenous nucleic acid may encode an antibody.

Alternatively, the protein may be useful for research purposes. Suitably the exogenous nucleic acid sequence may comprise one or more genes which encode one or more proteins which it is desirable to study in the cell. For example, the exogenous nucleic acid sequence may comprise one or more genes which encode one or more proteins of a metabolic or signalling pathway. Suitably the exogenous nucleic acid may comprise one or more genes which encode several, or in some cases all, of the proteins in a pathway.

In one embodiment, the exogenous nucleic acid may comprise several genes which form a genetic circuit. For example: Oscillators, Bistable switches, Logical operators, Analog tuners, and combinations thereof.

In one embodiment, the exogenous nucleic acid may encode one or more optogenic molecular sensors. Suitably the optogenic molecular sensors allow a biological pathway or mechanism to be visualised.

Suitable optogenic sensors comprise a sensing domain fused to a reporter domain, for example genetically encoded voltage indicators (GEVI} or genetically encoded calcium indicators (GECI). Suitable examples of GEVIs and GECls are known in the art. Examples of GEVIs include: VSFP2.3, Arclight, FlicR1, ASAP family (ASAP1, ASAP2s, ASAP3), Voltron family (Voltron525, Voltron635, Quasars, Archonl, and NIR-butterfly. Examples of GECÍs include: GCaMP family; R-CaMP family; R-GECO family; and NIR-GECO. In some embodiments, the GEV} and GECI may be fused, for example in the GEVI-GECI: CaViar.

Alternatively, optogenic sensors may include a membrane localised reporter. Suitably comprising a membrane localisation tag fused to a reporter domain. Suitable membrane localisation tags may comprise the membrane targeting sequences from the proteins Lek, Gap43, Src, Kras, and p63RhoGEF.

Suitably any reporter domain may be used, suitably any fluorescent reporter domain. Suitable examples of fluorescent reporters are described herein elsewhere. Suitably other reporter domains/proteins may be used for example tags. Suitable tags may be FLAG, His, or others known in the art.

In one embodiment, the exogenous nucleic acid may encode a GEVI, a GECI and a membrane localised reporter. In one embodiment, the exogenous nucleic acid may encode the GEVI ASAP2f; the GECI jRCaMP1b; and the fluorescent reporter miRFP703 fused to a membrane targeting sequence from the naturally occurring protein, Lck.

Inanother example, the exogenous nucleic acid sequence may comprise one or more genes which encode a protein that is known to cause a disease or disorder. Suitably to enable researchers to study the disease or disorder. Suitably, therefore, the exogenous nucleic acid sequence may comprise a variant of a gene. Suitably the variant gene may comprise one or more mutations relative to the wild type gene sequence. Suitably the or each mutation may be present in the coding sequence of the gene or in non-coding sequences of the gene. Suitably the variant gene may cause a disease or disorder, suitably the variant gene may encode a protein which causes a diseases or disorder. Suitably the protein may have an altered structure or altered expression which is different from the wild-type, suitably caused by the or each mutation. Alternatively, the variant gene may not be known to cause any disease or disorder, the variant gene may be created to test if the one or more mutations will cause a disease or disorder. Therefore, in one embodiment, the exogenous nucleic acid sequence may comprise a novel variant of a gene.

In one embodiment, the exogenous nucleic acid may comprise one or more variant genes encoding one or more proteins.

Suitably the exogenous nucleic acid comprising one or more variant genes may also comprise one or more reporter genes, suitably linked to each variant gene. Suitably this allows the resulting protein expressed from a variant gene to be visualised in the cell. Suitably the reporter genes may encode a reported domain or protein. Suitably the reporter domain or protein may be fluorescent or may be a tag as explained elsewhere. in a further embodiment where a plurality of cells are created by the method of the invention, for example in a population of cells, suitably each cell may comprise a different exogenous nucleic acid sequence.

Suitably inserted at the same location in the genome. Suitably this allows the study of different variants of the same gene within the same genetic context.

In one embodiment, each cell may comprise a different variant of a gene, suitably of the same gene. Alternatively each cell may comprise a different arrangement of a plurality of genes, for example a different order or combination of the same group of genes. Suitably therefore the invention may be used to create libraries of cells. Suitably each cell in the library may comprise a different exogenous nucleic acid sequence. Suitably each cell in the library may comprise a different variant of the same gene. Suitably each cell in the library may comprise a different order or combination of the same group of genes. Suitably such cell libraries may be used for screening, for example to screen candidate therapeutics.

Suitably the exogenous nucleic acid sequence may also comprise regulatory elements and/or non-coding sequences. Suitably regulatory elements include: promoters, enhancers, silencers, insulators, 3'UTRs, 5'UTRs, introns, inverted terminal repeats (ITRs), recombinase recognition sites, RNA regulatory sequence, kozak sequence, terminator (polyadenylation sequence), internal ribosome entry site {IRES), uORFs, and the like.

Suitably the exogenous nucleic acid sequence may also comprise short coding sequences. Suitably short coding sequences for linkers, cleavage sites, signal peptides, affinity tags and the like.

Suitably therefore, the exogenous nucleic acid sequence may comprise one or more genes, and additionally one or more regulatory elements and/or non-coding sequences and optionally one or more short coding sequences.

Suitably, the exogenous nucleic acid sequence may comprise upstream and downstream sequences surrounding a gene, for example the 5’ and 3’ sequences flanking a gene, in addition to the nucleic acid sequence encoding the gene. Suitably the upstream and downstream regions may encode regulatory elements and/or non-coding sequences and/or short coding sequences as above.

Advantageously, the inventors have found that the ability to integrate larger exogenous nucleic acid sequences means that not only a gene of interest can be inserted into the genome for expression in the cell, but also the surrounding upstream and downstream nucleic acid sequences containing various regulatory elements which control expression of the gene. This means that the genetic context of the gene can be retained after insertion into the genome of a cell leading to more accurate expression of the gene, and more accurate research.

Cell The present invention involves integration of a desired exogenous nucleic acid sequence into the genome ofacell.

Suitably the cell may be any cell. Suitably the cell may be prokaryotic or eukaryotic. Suitably the cell may be a plant cell, a bacterial cell, a fungal cell, or an animal cell. Suitably the cell may be a eukaryotic cell. Suitably the cell may be an animal cell. Suitably the cell may be a mammal, fish, reptile, insect, or bird cell. Suitably the cell may be a mammalian cell. Suitably the cell may be a human cell.

In some embodiments, the cell is a stem cell, suitably a human stem cell. Suitably the stem cell may be an adult or a fetal stem cell. Suitably the stem cell may be a hepatic stem cell, haematopoietic stem cell, adipose derived stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, and endothelial stem cell, a neural stem cell, an olfactory stem cell, or a neural crest stem cell.

Suitably the cell may be a human pluripotent stem cell. In some embodiments the cell may be a human embryonic stem cell. In some embodiments, the stem cell is a human induced pluripotent stem cell {hiPSC). in one embodiment, the hiPSC may have been induced or differentiated to form a certain cell type.in one embodiment, the cell may be a hiPSC-cardiomyocyte , hiPSC-neuron, hiPSC-hepatocyte, hiPSC-bone marrow cell, hiPSC-endothelial cell, for example. in one embodiment the cell may be a model cell for research, for example a HEK293 cel lin another preferred embodiment of the invention the cell may be a retinal cell, e.g. a retinal pigmented epithelium (RPE) cell, for example ARPE-19 (ATCC CRL-2302). Alternatively, a hamster cell line, e.g. BHK21, BHK TK-, CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1, CHO-S. Furthermore, murine myeloma cells, preferably NSO and Sp2/0 cells or the derivatives/progenies of any of such cell lines are also well-known as production cell lines for biopharmaceutical proteins. Further suitable cells include PER.C6; HT-1080; HuH-7; HKB-11, or CAP cells. Suitable host cells are commercially available, for example, from culture collections such as the DSMZ (Deutsche Sammlung von Mikroorganismen and Zeilkuituren GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

Particularly preferred cells are human embryonic kidney cells (especially, HEK-293 cells, and in particular HEK-293-F cells), CHO cells (particularly CHO-K1SV cells). it may be preferred that cells are established, adapted, and completely cultivated under serum free conditions, and optionally in media which are free of any protein/peptide of animal origin. Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma}, Minimal Essential Medium (MEM; Sigma}, Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA}, CHO-S-SFMit (Invtirogen), serum- free CHO Medium (Sigma), protein-free CHO Medium (Sigma), EX-CELL Media (SAFC), CDM4CHO and SFMA4CHO (HyClone) are exemplary appropriate nutrient solutions. Any of the media may be supplemented as necessary with a variety of compounds examples of which are hormones and/or other growth factors (such as insulin, transferrin, epidermal growth factor, insulin like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate}, buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics, trace elements. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. In the present invention the use of serum-free medium is preferred, but media supplemented with a suitable amount of serum can also be used for the cultivation of cells. For the growth and selection of genetically modified cells expressing a selectable gene a suitable selection agent is added to the culture medium.

In one embodiment, there is provided a cell culture comprising a population of cells of the present invention and medium sufficient to support growth of the cells.

The method of making a population of cells of the invention may comprise maintaining said population of cells under suitable conditions for proliferation of the cells. The cells may therefore be provided in suitable cell culture condition for the type of cell being used. Suitable cell culture conditions for the various cell types are well-known to the skilled person or can be readily identified from the literature. Suitably therefore the method may comprise a step of expanding the cells, suitably into a population of cells.

The methods can be carried out in any suitable reactor including but not limited to stirred tank, airlift, fibre, microfibre, hollow fibre, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors. As used herein, "reactor" can include a fermenter or fermentation unit, or any other reaction vessel and the term "reactor" is used interchangeably with fermenter”. For example, in some aspects, an example bioreactor unit can perform one or more, or all, of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and C02 levels, maintenance of pH level, agitation (e.g., stirring), and/or cleaning/sterilizing. Example reactor units, such as a fermentation unit, may contain multiple reactors within the unit, for example the unit can have 1 to 10 or more bioreactors in each unit. In various embodiments, the bioreactor can be suitable for batch, semi fed-batch, fed-batch, perfusion, and/or a continuous fermentation process. In some embodiments, the bioreactor can have a volume of from about 100 ml to about 50,000 litres, preferably 10 litres or higher. Additionally, suitable reactors can be multi-use, single-use, disposable, or non- disposable and can be formed of any suitable material. U.S. Publication Nos. 2013/0280797, 2012/0077429, 2011/0280797, 2009/0305626, and U.S. Patent Nos. 8,298,054, 7,629,167, and 5,656,491 (hereby incorporated by reference in their entirety) describe exemplary systems that may be used in the present invention.

In one embodiment, the invention provides a reactor vessel comprising a cell culture comprising cells according to the invention and a medium sufficient to support growth of the cell.

In another embodiment, the invention provides the use of vector or a cell according to the present invention in a bioprocessing method for the manufacture of a product of interest, e.g. a therapeutic product. Alternatively, the invention provides the use of vector or a cell according to the present invention in gene therapy, suitably gene therapy of a subject.

For example, the present invention provides a method for producing an expression product, the method comprising the steps of: (a) providing a population of cells comprising an exogenous nucleic sequence successfully integrated into the genome, preferably produced according to the method of the first aspect; (b) culturing said population of cells such that the exogenous nucleic acid is expressed and the expression product is produced; and (c) recovering the expression product.

The method is suitably a cell culture method. In some embodiments, the method is a method of bioprocessing, i.e. a process that uses living cells to obtain desired products. Preferred exogenous nucleic acid sequences are discussed herein. The expression product of the exogenous nucleic acid sequence may be useful for therapeutic, cosmetic, research or other industrial processes.

For example, the present invention provides a method of gene therapy of a subject, preferably a human, in need thereof, the method comprising: (a) Providing the subject with a cell comprising an exogenous nucleic sequence successfully integrated into the genome, preferably produced according to the method of the first aspect; wherein the exogenous nucleic acid encodes a therapeutic expression product; and (b} expressing a therapeutically effective amount of the therapeutic expression product in the subject.

Suitably the method of producing the cell may be carried out in vivo or in vitro, preferably in vitro before providing the subject with the cell.

Expression levels of the expression product {e.g. protein) can be measured by various conventional means, such as by antibody-based assays, e.g. a Western Blot or an ELISA assay, for instance to evaluate whether therapeutic expression of the expression product is achieved. Expression of the expression product may also be measured in a bioassay that detects an enzymatic or biological activity of the gene product.

The therapeutic product may have a therapeutic effect in any suitable location in the subject. For example, it may have an effect in the cells where it is expressed, in neighbouring cells or tissues, or it may be secreted and enter the bloodstream and treat a condition elsewhere in the body.

Suitable therapeutic gene/expression products i.e. proteins are discussed elsewhere herein.

in general, the subject in need thereof will be a mammal, and preferably primate, more preferably a human. Typically, the subject in need thereof will display symptoms characteristic of a disease. The method typically comprises ameliorating the symptoms displayed by the subject in need thereof, by expressing the therapeutic amount of the therapeutic expression product.

In one embodiment, the invention provides the cells or vectors according to the present invention or pharmaceutical compositions comprising said cells for use in a method of treatment or therapy. In one embodiment, the invention provides the cells or vectors according to the present invention for use in the manufacture of a pharmaceutical composition, optionally for use in a bioprocessing method for the manufacture of a product of interest, e.g. a therapeutic product.

The pharmaceutical composition may be formulated with a pharmaceutically acceptable excipient, i.e, one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilisers, etc. The pharmaceutical composition may be provided in the form of a kit. The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

In one embodiment, the cell may comprise more than one landing pad within its genome. Suitably the cell may comprise two landing pads within its genome. Suitably each landing pad comprises the structure as described herein. However suitably each landing pad is at a different location in the genome, and suitably each landing pad comprises different recombination sites to prevent incorrect insertion. Suitably in such an embodiment, step (a) of the method may comprise providing a cell with a first and second landing pad in its genome. Suitably the first landing pad may comprise a BxB1 recombination site and the second landing pad may comprise a PhiC31 recombination site, or vice versa. Suitably therefore the method may then comprise a step (b) of introducing more than one integration vector into the cell. Suitably a first integration vector and a second integration vector. Suitably a first integration vector for insertion into the first landing pad, and the second integration vector for insertion into the second landing pad. Suitably the or each integration vector has recombination sites matching those of the intended landing pad. Suitably,

for example, the first integration vector may have a BxB1 recombination site and the second integration vector may have a PhiC31 recombination site, or vice versa.

Selectable Marker and Promoter The present invention makes use of a selectable marker gene in the landing pad, which is only expressed when successful recombination occurs and the promoter and start codon is placed upstream and in-frame with the nucleic acid sequence encoding the selectable marker gene.

Suitably any selectable marker may be used. Suitably the selectable marker may be any nucleic acid sequence which allows a distinction to be made between celis that express it, and those that do not, using simple laboratory procedures. Suitably the selectable marker may be a gene encoding a visual reporter, antibiotic resistance, or a cell surface antigen for example.

Suitable visual reporters are known in the art but may include luciferase, beta-galactosidase, GFP, RFP, YFP, CFP, GUS, for example. Suitably the visual reporter is a fluorescent reporter. Suitable fluorescent reporters include GFP, RFP, YFP, CFP or far-RFP for example.

Suitable antibiotic resistance genes are known in the art but may encode resistance to one of the following: blasticidin, kanamycin, ampicillin, neomycin, bleomycin, chloramphenicol, hygromycin, mitomycin, puromycin, phleomycin, zeocin, geneticin, for example. In one embodiment, the antibiotic resistance gene encodes resistance to blasticidin or bleomycin. Suitably the blasticidin resistance gene is bsr, bls, or BSD. Suitably the bleomycin resistance gene is ble.

Suitable cell surface antigens that may be used as markers are known in the art by may include CD24, LNGFR, CD19, CD20, CD34, PTKR, CD4, H-2K¥, for example Suitably such cell surface antigens may be modified for use as markers.

Suitably the promoter is a promoter which drives expression of the selectable marker. Suitably the promoter is a constitutive promoter. Suitably so that expression of the selectable marker is constant when upstream and in-frame with the nucleic acid sequence encoding the selectable marker. Suitable constitutive promoters include: CMV, EF1A, SV40, PGK1, Ubc, human beta actin, CAG, TRE, Ac5, polyhedrin, CaMV, Ubi, TEF1, GS for example. In one embodiment the promoter is EF1A.

In one embodiment, the selectable marker gene is a gene encoding resistance to blasticidin or bleomycin.

Selection

The method of the invention may further comprise a step of selection. Suitably this may comprise identifying a cell expressing the selectable marker gene, and selecting the cell which expresses the selectable marker gene. Suitably expression of the selectable marker gene indicates that the recombination has been successful and that the integration vector is integrated into the genome of the cell in the correct position.

Suitably identifying the cells which express the selectable marker gene is carried out by exposing the cells which have undergone the method to a selection means. Suitably the selection means must match the selectable marker which has been used.

Suitably if the selectable marker is a visual reporter then no selection means may be necessary, and the visual reporter may be visible without any further steps. In some embodiments, the selection means may comprise exposing the cells to light, suitably light of a certain wavelength in order to observe the visual reporter. In some embodiments, the selection means may comprise exposing the cells to a chemical, which may induce the expression or activity of the visual reporter. In one embodiment, if the selectable marker is GFP then the cells are exposed to blue light. In one embodiment, if the selectable marker is luciferase, then the cells are exposed to the substrate luciferin.

Suitably if the selectable marker is an antibiotic resistance gene, then the selection means may comprise exposing the cells to an antibiotic. Suitably to the antibiotic for which the gene is intended to provide resistance. In one embodiment, if the selectable marker is a blasticidin antibiotic resistance gene, then the cells are exposed to blasticidin.

Suitably if the selectable marker is a cell surface antigen, then the selection means may comprise exposing the cells to an antibody. Suitably to an antibody which binds to the cell surface antigen. Suitably the antibody may be labelled so as to observe the antibody binding. In one embodiment, if the selectable marker is H-2K¥, then the cells are exposed to mouse antibody Anti-H-2H-FITS. This system may suitably be available from Miltenyi Biotech, the ‘Macselect KK System’.

Once the cells which express the selectable marker have been identified they may then be selected. Suitably selecting the cells comprises a step of selecting the cells that have been identified to express the selectable marker.

Suitably if the selectable marker is a visual reporter then selecting the cells may comprise selecting those cells which fluoresce or luminesce. In one embodiment, if the selectable marker is GFP then selecting the cells comprises selecting the cells which fluoresce green when exposed to blue light.

Suitably if the selectable marker is antibiotic resistance gene then selecting the cells may comprise selecting those cells which survive exposure to the antibiotic.

Suitably those that survive on or in a medium comprising the antibiotic.

Suitably if the selectable marker is a cell surface marker, then the selecting the cells may comprise selecting those cells to which the antibody has bound.

In addition, the selection step may further comprise confirming that the integration vector has integrated into the genome by molecular methods, for example by PCR or by sequencing.

Suitably PCR may be used to detect the formation of a recombination scar in the genome of the cell.

Suitably PCR may be used to detect formation of the recombination scars attR and/or attL in the genome of the cell.

Suitably the selection step may further comprise isolating the cells that have been identified to express the selectable marker, for example by harvesting the cells.

Suitably the selected cells may then be further cultured and/or passaged.

Suitably the method may comprise a step of enriching the cells, suitably enriching the selected cells that express the selectable marker.

Suitably a step of enriching the cells comprises culturing the selected cells, optionally in the presence of an antibiotic in embodiments where the selectable marker is an antibiotic resistance gene.

Suitably a step of enriching the cells comprises expanding the selected cells.

Optionally, a step of enriching the cells may comprise further rounds of selection, suitably using the selectable marker.

Suitably the selected cells may then be purified.

Suitably the method may comprise a step of purifying the selected cells that express the selectable marker.

Introduction of Nucleic Acid Sequences The method of the invention comprises a step of introducing the integration vector into the cell.

Furthermore, the method comprises at least one recombination step which is catalysed by a recombinase enzyme.

As discussed above, the or each recombinase enzyme is also introduced into the cell.

Suitably the or each recombinase enzyme may be introduced into the cell as a nucleic acid sequence encoding the or each recombinase, which may be comprised upon a vector, or which may be introduced as mRNA or directly as a protein.

The vectors used in the method of the invention may be any suitable known vector for carrying exogenous nucleic acid sequences into a cell.

The vector can be any naturally occurring or synthetically generated constructs suitable for uptake, proliferation, expression or transmission of nucleic acids in a cell, e.g. plasmids, minicircles, phagemids,

cosmids, artificial chromosomes/mini-chromosomes such as BACs or YACs, bacteriophages, viruses such as baculovirus, retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, or bacteriophages. Methods for the construction of vectors are well known to the person skilled in the art, and they are described in various publications and reference texts. In particular, techniques for constructing suitable vectors, including a description of the functional and regulatory components such as promoters, enhancers, termination and polyadenylation signals, selection markers, origins of replication, and splicing signals, are known to the person skilled in the art.

In preferred embodiments, the vector may be a eukaryotic expression vector. Eukaryotic expression vectors will typically contain also prokaryotic sequences that facilitate the propagation of the vector in bacteria such as an origin of replication and antibiotic resistance genes for selection in bacteria. A variety of eukaryotic expression vectors, containing a cloning site into which a polynucleotide can be operably linked, are well known in the art and several are commercially available from companies such as Stratagene, La Jolla, CA; Invitrogen, Carlsbad, CA; and Promega, Madison, Wi.

in some embodiments of the invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, plRES2-DsRed2, pDsRed2-Mito, pCMV-EGFP available from Clontech. Many other vectors are well-known and commercially available. For mammalian cells adenoviral vectors, the pSV and the pCMV series of vectors are particularly well-known non-limiting examples. There are many well-known yeast expression vectors including, without limitation, yeast integrative plasmids (Yip) and yeast replicative plasmids (YRp). For plants the Ti plasmid of agrobacterium is an exemplary expression vector, and plant viruses also provide suitable expression vectors, e.g. tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

In some embodiments of the invention, the vector is a plasmid. Such a plasmid may include a variety of other functional nucleic acid sequences, as described herein, such as one or more origins of replication, polycloning sites and the like.

The vector may be a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector. In some preferred embodiments the vector is an AAV vector. In some preferred embodiments the AAV has a serotype suitable for transduction of a particular cell or tissue type. In some embodiments, the AAV is selected from the group consisting of: AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, or derivatives thereof. AAV vectors are suitably used as self-complementary, double-stranded AAV vectors (scAAV} in order to overcome one of the limiting steps in AAV transduction (i.e. single-stranded to double-stranded AAV conversion), although the use of single-stranded AAV vectors (ssAAV) is also encompassed herein. In some embodiments of the invention, the AAV vector is chimeric, meaning it comprises components from atleast two AAV serotypes, such as the ITRs of an AAV2 and the capsid protein of an AAV5.

In one embodiment, there is provided a recombinant virion (viral particle} comprising a viral vector. The virion can be produced using conventional techniques known to the skilled person.

Suitable plasmids include pUC, pBR322, pET, pGEX, pColE1, pACYC, pBluescript, pGEM, TOPO, for example. Suitable BACs include: pBeloBAC11, pBACe3.6, pPAC4, pCCl1BAC, pSMART VC for example,. Suitable cosmids include: pWEB-TNC, SuperCos 1, pFos1, for example. Suitable circular YACs include pTARBAC for example. Suitable circular HACs include: tet-O HAC, BAC: HAC, for example.

In one embodiment, the vectors used in the present invention are plasmids.

In some embodiments the plasmids used for each element of the invention, such as the integration vector, the landing pad vector, the vector comprising the first recombinase, and the vector comprising the second recombinase are those described in full in the examples herein.

Suitable methods for introducing a nucleic acid sequence such as a vector into a cell are known in the art. Suitably the nucleic acid sequences used in the present invention may be introduced into the cell by transfection, or transduction.

Suitable methods of transfection may be physical or chemical. Suitable physical methods include microinjection, gene gun, impalefection, hydrostatic pressure, sonication, optical transfection, protoplast fusion and electroporation. Suitable chemical methods include lipofection, or the use of polymers, nanoparticles, dendrimers, or calcium phosphate.

In one embodiment, the vectors or nucleic acid sequences are transfected into the cell by electroporation or lipofection.

Suitable methods of transduction include viral transduction. Suitably viral transduction may use prokaryotic viruses such as phage as vectors, or replication deficient eukaryotic viruses such as lentivirus, retrovirus, or AAV as vectors.

In one embodiment, the vectors are replication deficient viral vectors introduced into the cell by transduction.

Suitably for the introduction of proteins into the cell, for example in the embodiments where the or each recombinase, or Cas endonuclease may be introduced as a protein, this is carried out by lipofection, electroporation or iTOP (as described in PCT/IB2014/064127).

FIGURES Figure 1 shows: Generation of hiPSC acceptor lines for Bxb1- or phiC31-mediated integration (A) Schematic of the landing pad (LP) cassettes specifically recognised by either Bxb1 (left) or phiC31 (C31; right) integrases and strategy for targeting these cassettes to the PPP1R12C locus. The large black arrows represent the pGK promoter driving constitutive expression of the fluorescent reporter genes, eGFP or mCherry. The * indicates that the antibiotic positive selection marker lacks an ATG initiation codon and so is not expressed in the resulting targeted hiPSCs. Half arrows indicate primer binding sites with dotted lines representing the resulting amplicons generated by PCR {B} Fluorescence images (top) and flow cytometry plots (below) indicating expression of the eGFP and mCherry reporters in the resulting targeted AAVS1-acceptor hiPSC lines. Scale bar, 50 um (C} PCR amplification, using primer pairs indicated in panel A, of genomic DNA confirming specific targeting of each of the LP cassettes depending on the resulting AAVS1-acceptor hiPSC line screened. Also, that only one allele of the PPPIR12C locus was targeted in the AAVS1-Bxb1 and AAVS1-pC31 hiPSC lines, while both alleles were targeted in the AAVS1-dual hiPSCs (D} ddPCR confirming that each AAVS1-acceptor hiPSC line had either O or 1 copy of each of the LP cassettes inserted into the genomic DNA; Figure 2 shows: Bxb1 mediates efficient targeted integration of donor constructs into hiPSCs without size restrictions (A) Schematic of procedure for integrating donor constructs by either Bxb1 (/eft) or PhiC31 (right) integrases into the corresponding LP cassette. Correct integration of the donor construct results in the insertion of the Efla promoter and an ATG initiation codon upstream and in-frame with the antibiotic positive selection marker present in the LP cassette and expression of the gene. “Payload” indicates region in the donor construct where DNA sequences to integrate are inserted. Half arrows indicate primer binding sites with dotted lines representing the resulting amplicons generated by PCR {B} PCR amplification, using primer pairs indicated in panel A, of genomic DNA confirming integration of each donor construct into their respective LP cassette. “Pre” indicates prior to, and “Post” following antibiotic selection. PCR amplification using a primer pair that flanks the attP target sites within the LP cassette indicated that hiPSCs in which the donor construct did not integrate remained after antibiotic selection, {C) Alkaline phosphatase (AP) staining of hiPSCs following antibiotic selection of cells transfected with plasmids expressing either Bxb1 or Phi C31 integrase as well as the Bxb1- or Phi C31- donor constructs demonstrate that integration is highly specific for each integrase and selection cassette.

AP staining also indicates that Bxb1 more efficiently integrates constructs than Phi C31 (D) Schematic illustrating the location of the primers and probes used in the ddPCR assay to quantify the percentage of cells in which Bxb1-donor constructs had integrated following antibiotic selection (E) Representative one-dimensional plots to quantify the efficiency of integration donor constructs containing payloads ranging from 0 — 50 kb into the Bxb1-LP.

Droplets containing amplicons generated from cells in which integration did not occur (light blue dots) resulted in one fluorescent probe binding, while two fluorescent probes could anneal to amplicons generated from recombined hiPSCs (dark blue dots} and resulting in an increase in fluorescence intensity.

Integration efficiency (%) was determined by counting the number of droplets in which 2 fluorescent probes had annealed divided by the total number of droplets containing either amplicon.

Transfection of a Phi C31-donor construct into hiPSCs containing the Bxb1-LP resulted in no integration, confirming the specificity of the system (F} Average percentage of recombined hiPSCs for each of the indicated Bxb1-donor constructs following a single round of blasticidin selection (n=4 independent transfections). Error bars represent SEM; Figure 3 shows: Integration of BAC donor construct into AAVS1-acc or AAVS1-Bxb1 hiPSCs using the method of invention {A) Diagram of the Bxb1-BAC donor construct indicating the 3 genes (AOC1, KCNH2, NOS3) encoded by the BAC, as well as modifications made to the BAC for use as a donor construct {B} Schematic of procedure for integrating the BAC_attB(bxb) vector by Bxb1 into the AAVS1-acc locus.

Correct integration of the donor BAC vector results in the insertion of the Efla promoter and an ATG initiation codon upstream and in-frame with the blasticidin resistance gene lacking these features leading to expression of the gene. half arrows, primer binding sites; dotted lines, resulting amplicons generated by PCR (C} PCR amplification, using primer pairs indicated in panel B, of genomic DNA confirming integration of the Bxb1-BAC donor vector into hiPSCs containing the Bxb1-LP. (D) ddPCR confirming that a hiPSC clone identified as having the BAC_attB(bxb) donor integrated into the Bxb1-LP {+ BAC_attB(bxb}} contained a third copy of the genes AOC1, KCNH2, and NOS3. The non-integrated

AAVSI-Bxbl (- BAC _attB(bxb)} hiPSC line retains 2 copies of each.

Both lines contain a single copy of the LP cassette transgenes, eGFP and BsdR Figure 4 shows: Cre recombinase-mediated excision of auxiliary sequences following donor vector integration (A) Schematic of procedure for excising by cre recombinase the positive selection cassettes as well as the vector backbone following integration of the donor vector into the Bxb1-LP.

Dashed lines indicate the sequences excised.

The resulting modified locus comprises of only the introduced DNA payload and a single copy of the lox sequences, loxP and /oxP257. Half arrows indicate primer binding sites with dotted lines representing the resulting amplicons generated by PCR (B) PCR amplification,

using primer pairs indicated in panel A, of genomic DNA confirming excision of the /ox-flanked (floxed) sequences upon expression of cre recombinase (+) (C) Percentage of resulting hiPSC clones in which both floxed sequences are excised following expression of cre recombinase.

Graph indicates the overall average % efficiency, as well that efficiency of excision is not influenced by the size of the DNA payload inserted.

Error bars represent SEM (D} Long-range PCR amplification of the entire payload (2 kb — 15 kb) inserted, confirming that no fragments of the payload were lost while performing the method of invention; Figure 5 shows: Assessment of individual genetically-encoded fluorescent reporters introduced using the method of the invention for evaluating excitation, Ca? transients and contractility kinetics in hiPSC- CMs (A) Diagrams of the three donor vectors for integrating expression constructs for ASAP2T (left), jRCaMP1b (middie) and miRFP703 (right) using the method of invention (B} Schematic showing overall configuration of the AAVS1-targeted locus following the introduction of the fluorescent reporter with STRAIGHT-IN.

The resulting modified locus (AAVS1-reporter) comprised of the fluorescent reporter (ASAP2f, jRCaMP1b, miRFP703) downstream of a pCAG promoter and upstream of a polyA terminator signal.

For reporters that required it, a subcellular localisation signal was included at the 5’ end of the coding sequence.

Following expression of cre recombinase and a single copy of the loxP and /oxP257 sequences flanked the expression construct.

Half arrows indicate primer binding sites with dotted lines representing the resulting amplicons generated by PCR.

The size of the resulting amplicons for each reporter are noted in panel C {C} PCR amplification of genomic DNA confirming targeted integration of each of the fluorescent reporters into the AAVS1 locus using STRAIGHT-IN {D} Fluorescence images of the AAVS1-reporter hiPSCs showing expression and cellular localisation of the reporters indicated above each image.

Scale bars, 50 um {E) Flow cytometry plots indicating percentage and level of expression of each of the reporters in the corresponding AAVS1-reporter hiPSC line (F} Schematic of protocol to differentiate the AAVS1-reporter hiPSC lines to cardiomyocytes. mBEL, modified low insulin-BPEL medium (G} Immunofluorescence images of the cardiac sarcomeric protein a-actinin indicating each of the AAVS1-reporter hiPSC lines could differentiate to cardiomyocytes.

Images on the right are magnifications of the regions within the dotted boxes.

Scale bars, 25 um {H) Fluorescence images of cardiomyocytes differentiated from each of AAVSI-reporter hiPSCs showing expression and expected cellular localisation of the reporters.

Scale bars, 50 um (1) Flow cytometry plots indicating percentage and level of expression of each of the reporters in the cardiomyocytes differentiated from each of the corresponding AAVS1-reporter hiPSC lines (J) Representative averaged time plots of baseline-normalized fluorescence signals of cardiomyocytes from the indicated AAVS1-reporter hiPSC lines stimulated at 1.2 Hz.

The AP trace is shown in green, cytosolic Ca? flux in red, and contraction-relaxation in orange;

Figure 6 shows: Generation of a multi-parameter reporter hiPSC line using the method of the invention for simultaneously evaluating the EC-coupling cascade in hiPSC-CMs (A) Diagram of the donor vector for integrating two expression constructs for ASAP2f and single expression constructs for jRCaMP1b and miRFP703 using the method of the invention {B} One-dimensional plot quantifying the integration efficiency of the Bxb1-AJMA donor construct into the Bxb1-acceptor hiPSCs after one round of blasticidin selection.

Dark blue dots indicate droplets containing the amplicon obtained following integration, while light blue dots indicate droplets containing the amplicon generated if no integration has occurred.

The percentage denotes the calculated integration efficiency {C) Schematic of the resulting AAVS1-AIMA locus (top). Half arrows, primer binding sites; dotted lines, the resulting amplicons with the expected size indicated in brackets.

PCR screening (bottom), using the primer pairs indicated, confirming targeted integration of the multi-reporter construct and no internal rearrangement of the transgenes.

A DNA ladder was loaded in the first column of the gel, with the size of selected bands indicated (D} Schematic showing overall configuration of the AAVSI-targeted locus following the introduction of a DNA construct comprising of one or two copies of all three fluorescent reporters using the method invention, and overall size of the DNA payload integrated {E) Fluorescence images of the AAVS1-AJMA hiPSC line showing expression and cellular localisation of each of the reporters integrated.

Scale bars, 75 um (F) Flow cytometry plots indicating percentage and level of expression of each of the reporters in the AAVS1-AJMA hiPSC line {G) Fluorescence images of AAVSI- AIMA hiPSC-CMs confirming expression and expected cellular localisation of the reporters {MH} Flow cytometry plots indicating percentage and level of expression of each of the reporters in the AAVSI- AIMA hiPSC-CMs (1) Representative time plots of baseline-normalized fluorescence signals from the AAVS1-AJMA hiPSC-CMs stimulated at 1.2 Hz.

The action potential signal (ASAP2f, green), cytosolic Ca?* flux (jRCaMP1b, red), and contraction-relaxation (miRFP703, orange) were obtained from the same ROI {J} Representative action potentials of AAVSI-AJMA hiPSC-CMs in response to vehicle (DMSO) and 100 nM E-4031 (left) and average APDso (middle) and APDso (right) values as determined by changes in the ASAP2f fluorescent signal (K) Representative cytosolic Ca?’ flux signals of AAVS1-AJMA hiPSC-CMs in response to vehicle (DMSO) and 25 nM verapamil (left) and average amplitude (middle) and Ca2+ duration at 50% (CaDso; right} values as determined by changes in the jJRCaMP1b fluorescent signal (L) Representative contraction-relaxation signals of AAVS1-AJMA hiPSC-CMs in response to vehicle (DMSO)

and 100 nM isoprenaline (left) and average peak-to-peak time (middle) and contraction duration at 90% (CoDso; right) values as determined by changes in the displacement of the miRFP703 fluorescent signal; Figure 7 shows: Employing the method of the invention to generate a panel of isogenic hiPSC lines of cardiac disease-associated KCNH2 variants as well as corresponding wild-type (A) Schematic of the procedure to enable targeted heterozygous modification to a 50.6 kb genomic region that includes the gene, KCNH2. Half arrows indicate primer binding sites with dotted lines representing the resulting amplicons generated by PCR (B) PCR amplification, using primer pairs indicated in panel A, of genomic DNA confirming replacement of the KCNH2 gene with the Bxb1-LP (KCNH27A°), followed by re- introduction of the KCNH2 wildtype genomic sequence (KCNH2A<%), Auxiliary sequences detected upon integration (+) were excised following Cre recombinase expression (-). The size of the amplicons are indicated (C) ddPCR confirming that the KCNH27/A< hiPSCs and the resulting KCNH274<" hiPSC line after performing the procedure both contained the expected number of copies of KCNH2, it's adjacent genes NOS3 and AOC1, and the Bxb1-LP cassette transgenes, eGFP and BsdR {D} Overview of the procedure for simultaneously generating and identifying isogenic hiPSC clones for different KCNH2 variants, along with the approximate time required for each step {E) One-dimensional plot quantifying integration of the pool of Bxb1-KCNH2"*" donor constructs into the KCNH2*/A hiPSCs after 3 passages with blasticidin selection. Dark blue dots indicate droplets containing the amplicon obtained following integration, while light blue dots indicate droplets containing the amplicon generated if no integration has occurred. The percentage denotes the calculated integration efficiency {F) Overview of the genomic sequence and location within the KCNH2 gene of the introduced variant A561T, and sequence analysis from one of the resulting KCNH2"74<45617 hipSC clones indicating the heterozygous introduction of c.G1681A (G) Average FPD values confirming significant prolongation in cardiomyocytes generated from the KCNH2*/A<A561T hipSCs compared to KCNH2*/AcWt hiPSC-CMs. Values (n) refer to the number of measurements analyzed. Error bars represent SEM; *P < 0.0001; unpaired t-test; Figure 8 shows: Detection and demultiplexing of the KCNH2-variant lines generated by the method of invention. Overview of the genomic sequence and location (left), detection by ddPCR prior to (middle), and sequence analysis after demultiplexing (right) of the indicated KCNH2 variants introduced by STRAIGHT-IN (A558P (A); AS61T (B}; H578P (C); G584S (D); N588D (E}; N588K (F); A614V (G}; T618I (H); Te18S (1); F627L (J); G628S (K); N633S (L)). Note, while the KCNH2 variant, F627L, was integrated in the pool of transfected hiPSCs, clonal lines for this variant were not recovered following demultiplexing hence why no chromatogram is available; Figure 9 shows: Schematics of examples of integration vectors used with the method of invention. {A) The Bxb1 donor vectors used for inserting the DNA payloads and their respective sizes (kilobases, kb).

The pBR_attB(bxb)_lox plasmid (/eft} was used for cloning payloads <20 kb, either by enzymatic digestion and ligation using restriction enzymes indicated within the multi-cloning site (MCS), or by recombineering. The p15_attB(bxb)_lox plasmid (middle) was used for cloning payloads between ~20-50 kb by recombineering. The pBR_attB(bxb)_ccdB_lox plasmid (right) was used for cloning payloads via modular assembly strategies involving digestion of the plasmid with the type IIS restriction enzyme, Bpil. Half arrows, recombineering primers used to amplify the cloning vector with homology arms (HA) to the DNA payload attached; {B} Overview of the phiC31 donor vector; (C} Overview of the Bxb1 donor vectors containing DNA payloads between 72 — 50 kb, and the resulting size of the vector.

Figure 10 shows: Percentage of integrated cells generated using the method of invention increases in subsequent passages (P) of the cells if the cells are maintained in culture medium containing blasticidin. Figure 11 shows: PhiC31 integrase mediates efficient targeted integration of a donor construct into hiPSCs (A) One-dimensional plot quantifying the percentage of integrated hiPSCs cells with the donor vector prior (left) and post (right) zeocin selection. Orange dots indicate droplets containing the amplicon obtained following integration, while yellow dots indicate droplets containing the amplicon generated if no integration has occurred. The percentage denotes the calculated integration efficiency {B) Average percentage of recombined hiPSCs for the indicated PhiC31 donor vector following a single round of zeocin selection (n=3 independent transfections). The Bxb1-donor construct served as a control. Error bars represent SEM {C) Schematic of procedure for excising by fipo recombinase the positive selection cassettes as well as the vector backbone following integration of the donor vector into the PhiC31-LP. Dashed lines indicate the sequences excised. The resulting modified locus comprises of only the introduced DNA payload and a single copy of the FRT sequences, FRT and F3. Half arrows indicate primer binding sites with dotted lines representing the resulting amplicons generated by PCR {D} PCR amplification, using primer pairs indicated in panel C, of genomic DNA confirming excision of the FRT-flanked sequences upon expression of Fipo recombinase (+) (E) Percentage of resulting hiPSC clones in which both FRT-flanked sequences are excised following expression of Flpo recombinase; Figure 12 shows: Targeting of a genomic locus with an integration vector which contains also the promoter for driving expression of a selectable marker gene, a start codon, and a nucleic acid sequence encoding the selectable marker gene (BsdR) A) Schematic of procedure for integrating the indicated construct into the NR2F2 locus mediated by a Cas9-gRNA introduced as a protein complex. The selectable marker is expressed regardless of whether the construct is correctly integrated on not due to the presence of a constitutive promoter (large black arrow} and a start codon (ATG). Correct integration of the construct is determined by PCR screening. Small labelled arrows indicate primer binding sites (B) Schematic of the selection procedure to enrich for cells containing the targeted integrated construct.

The time required for each step is indicated (C) Average percentage of hiPSCs in which the construct has been integrated into the desired genomic locus (targeted), after 9 days of blasticidin selection (n=4 independent transfections). Error bars represent SEM;

Figure 13 shows: a schematic diagram of the recombination method of the invention including the landing pad, integration vector and resulting construct within the genome of a cell.

EXAMPLES Materials and Methods Sequences of oligonucleotides used for cloning and screening are listed in Tables 1 and 2. Table 1. Sequences of oligonucleotides and gBlocks used for vector construction lox251_pENTR_HiFi_Fwd ataacttcgtataggagactttatacgaagttaatggATCCCCTATAGTGAG | pENTR-eGFP-attP{bxb}-*bsdR ee PENTR loxP_HiFi_Fwd cgacgttgtaaaacgacggccagtcGCTAGCGAGCTCGGCGCG (SEQ pPENTR-eGFP-attP{bxb}-*bsdR ee PGK_GFP_HiFi_Fwd tttcgacctgcaggccaccATGGTGAGCAAGGGCGAG (SEQ ID NO PENTR-eGFP-attP{bxb}-*bsdR Tn [m— mm bxb_GFP_HiFi_Rev gacaaaccacgtatatctAGACATGATAAGATACATTGATGAGTTTG | pENTR-eGFP-attP{bxb)-*bsdR CT mE SV40_bxb_HiFi_Fwd tcttatcatgtctagatatacgtggtttgtctggtcaaccaccgcggtctcagtggtg | pENTR-eGFP-attP{bxb)-*bsdR tacggtacaaacccatatGCCAAGCCTTTGTCTCAAG (SEQ ID NO 16) lox251_bpA_HiFi_Rev tgtaatacgactcactataggggatccattaacttcgtataaagtctcctatacga PENTR-eGFP-attP{bxb}-*bsdR ee lox_eGFP_attP(Bxb)_lox25 | ctccaccccacagtggggcaAGTCACGACGTTGTAAAACGACG (SEQ | AAVS1-Bxb1-LP-TC ee lox_eGFP_attP{(Bxb} lox25 | accaatcctgtccctagtaATGACCATGTAATACGACTCACTATAGG AAVS1-Bxb1-LP-TC eea bxb_attB_lox gBlock CCATTTCAGGTGTCGTGAGGAATTCGCCACCATGGCCGGCTTG | pBR-bxb_attB donor

TCGACGACGGCGGTCTCCGTCGTCAGGATCATCCGGATCCATA ACTTCGTATAGCATACATTATACGAAGTTATCATGATATTCGGC AAGCAGGCATCGACTAGTTAATTAAGETAGCGCTGCAAGAAC

TCTTCCTCACGATAACTTCGTATAGGAGACTTTATACGAAGTTA AGCGCTCACTGGCCGTCGTTTTACA (SEQ 1D NO 20) pENTR-mCherry-attP{C31)- ee bleo_FRT-pENTR_HiFi_Fwd AGCTGATATCCCC (SEQ ID NO 22) bleoR*

cgacgttgtaaaacgacggccagtcTTAATTAAGAAGTTCCTATTCCGA | pENTR-mCherry-attP(C31)- PENTR_FRT_Hifibuild_Fwd AGTTC {SEQ ID NO 23) bleoR* pPENTR-mCherry-attP{C31)- PGK_cherry_HiFi_Rev tcaccatggtggcaattGTCGAAAGGCCCGGAGAT {SEQ iD NO 24) bleoR* eoR cgggectticgacaattgccaccATGGTGAGCAAGGGCGAG (SEQ ID | pENTR-mCherry-attP{C31)- PGK _cherry_HiFi_fFwd NO 25) bleoR* PENTR-mCherry-attP(C31)- C31_cherry_HiFi_ Rev gttggggcactacggatCCATAGAGCCCACCGCA {SEQ ID NO 26) bleoR* eoR tgggctctatggatccgtagtgccccaactggggtaaccitigagttctctcaegttg | pENTR-mCherry-attP{C31)- C31_bleo_HiFibuild_F wd geggcgtaggcgcGCCAAGTTGACCAGTGCC (SEQ ID NO 27) bleoR* cactataggggatatcagetggatgticgaagticctatactattigaagaatagg | pENTR-mCherry-attP{C31)- pENTR_F3_HiFi_Rev aacttcggaataggaacttcATAAGATACATTGATGAGTTTGGACAA | bleoR* AC {SEQ ID NO 28) FRT_pENTR_mCherry_attP | ctccaccccacagtggggcaAGTCACGACGTTGTAAAACGACG AAVS1-pC31-LP-TC (pC31}_F3_Fwd {SEQ ID NO 29) FRT_pENTR_mCherry_attP | accaatcctgtccctagtaATGACCATGTAATACGACTCACTATAGG AAVS1-C31-LP-TC (pC31}_F3_Rev G (SEQ ID NO 30) C31_attB_Fwd ACTCCACCTCACCCATCGGATC (SEQ ID NO 31) DEFBOS-attB{pC31)_FRT BamHi_puroRev153 ggatccGTGAGGAAGAGTTCTTGCAGC (SEQ ID NO 32) pEFBOS-attB{(¢bC31) FRT TGTAAAACGACGGCCAGTGAGCGCTgaagttcctatactattigaaG AATAGGAACTTCGgaataggaacttcGGATCCGATGGGTGAGGTG C31_attB_F3 gBlock GAGTACGCGCCCGGGGAGCCCAAGGGCACGCCCTGGCACCC GCACCGCGGCTTCGAGgcCATggTggcGAATTCCTCACGACACC TGAAATGG (SEQ ID NO 33) pPEFBOS-attB{pC31} FRT ACTCCACCTCACCCATCGGATCCgaagttcctattccgaagttcctattct FRT gBlock ctagaaagtataggaacttcatgatattcggcaagcaggcatcgACTAGTTAA TTAAGCTAGCgctgcaagaactcttcctcacggatcc (SEQ ID NO 34) pPEFBOS-attB{pC31} FRT pENTR-mCherry-attP(C31)- pPENTR_HiFi_rev GACTGGCCGTCGTTTTAC {SEQ ID NO 35) bleoR* gaagttcctattccgaagttcctattcttcaaatagtataggaacttcgaaCATCC | pENTR-mCherry-attP{C31)- bleo_FRT-pENTR_HiFi_Fwd AGCTGATATCCCC (SEQ ID NO 36) bleoR* p15-SV40 HiFi Fwd ATGGTTGCTGACTAATTGAGATGCAgcgctagcggagtgtatactg p15-bxb_attB donor {SEQ ID NO 37) amp-lox251 HiFi Rev TAATAGCGAAGAGGCCCGCACCGATacgtcaggtggcacttttcg p15-bxb_attB donor {SEQ ID NO 38) Ndel-Bpil-EFla_Fwd catatgaagacaatgccATTGGCTCCGGTGCCCGTC (SEQ ID NO pBR-attB{bxb)_ccdB_lox 39)

Ndei-Bpil_neod_Rev catatgaagacaagctaCGATGCCTGCTTGCCGAATATCATG (SEQ | pBR-attB(bxb)_ccdB_lox

ID NO 40) Xhol-Bpil-Esp3!_lacZ_Fwd | ctcgagaagacaatagcagagACGACAGGTTTCCCGACTGGAAAGC | pBR-attB({bxb)_ccdB_lox ET mea Xhoi-Bpil-Esp3i-lacZ_Rev ctcgagaagacaactccagagacGGTGTCGGGGCTGGCTTAAC (SEQ | pBR-attB{bxb}_ccdB_lox en Xhol-Bpit_purol53 Fwd ctcgagaagacaaggaGCTGCAAGAACTCTTCCTCACG (SEQ ID pBR-attB{bxb)_ccdB_lox rm ammmm— Hpal-Esp3l-Bpil-ccdB_Fwd | gttaacgtctcctagctgcctagtcttcACACATAACCAGGAGGTCAG pBR-attB(bxb}_ccd8_lox ee Hpal-Esp3i-Bpif-ccdB_Rev gttaacgtctcactecteccatgtcttcTGAAGTCAGCCCCATACGAT pBR-attB{bxb}_ccdB_lox ee EE BAC_EFla_recom_fwd catccgatgcaagtgtgtcgctgtcgacggtgaccctatagtcgagggacCTTA BAC _attB{bxb) TT een BAC_SV40_recom_Rev gctccgagaacgggtgcgcatagaaattgcatcaacgcatatagcgctagGATC | BAC attB(bxb) CT iran KCNH2_2kb_recom-Fwd aagatgctgatgactatgaataataaataattatcctgaggagaactccaGCTG | 2-15 kb DNA payload donors TT ensen KCNH2_2kb_recom-Rev gtagcagctgcaggacagtggccatgtctgcactcagccgggtctccagcCGAT | 2 kb DNA payload donor eae KCNH2_5kb_recom-Rev aggtgcaggcagatgtcagectgeaggeactcagggaageccticagecacCGA | 5 kb DNA payload donor RR =o il KCNH2_10kb_recom-Rev ctgggccgcagagcccctgtcctgctcgccttcccggetggggccgccatCGAT 10 kb DNA payload donor emmae | KCNH2_15kb_recom-Rev catcctcgttcttcacgggcaccacatccaccagacataggaagcagctccCGA | 15 kb DNA payload donor ee KCNH2_50kb_recom_Fwd | ctagcacctggaggigtgtgaggggccaggatggactgcggatagaccagtCGC | p15-attB_KCNH2_wt_donor nes KCNH2_50kb_recom_Rev aagggggcagggccigtcagatggatccctgacaaccatccgtctcaagtCGAT | pl5-attB_KCNH2_wt_donor see KCNH2_80b_M13_Fwd aggtgccctgtcttgggctctgaaggccctacatacaaagtcatcctatctgctga KCNH2-Bxb1-LP-TC ggcaaagatataaggttacctcg TGTAAAACGACGGCCAGTC {SEQ ID

NO 56) KCNH2_80bp_lox251_Rev | catttacatctgggccacagtgtccctccgtetccctetggccccggagcacatggc | KCNH2-Bxb1-LP-TC catctggtigtgctgactgtgctgCTATAGGGGATCCATTAACTTCG

(SEQ ID NO 57) KCNH2_ex7-amp_rec_Fwd | cttgccccccttgccccatcaacggaatgtgcccctteectgtcccccagCTAGC BAC with ccdB-amp cassette | remem

KCNH2_ex7-ccdB_rec_Rev | tggcccggctageagectcagittcctecaacttgggticctecaccgtgAGCCC | BAC with ccdB-amp cassette CATACGATATAAGTTG {SEQ ID NO 59) KCNH2 _ex7_ Fwd CTTGCCCCCCTTGCCCCATC (SEQ ID NO 60) p15- attB_KCNH2_variant_donors KCNH2_ex7_Rev TGGCCCGGCTAGCAGCCTC (SEQ ID NO 61) p15- attB_KCNH2_variant_donors Table 2. Sequences of oligonucleotides used for genotyping PCRs eee forward Sequence Reverse | Purpose DD me le

ENG NO [62 CCATGATATAGACGTTGTGGC | 63 | AAVS1 acc (Bxbl) targeting — 5' junction CCGGAACTCTGCCCTCTAA

TGTTG CGGCAGTTGGGATTCGTGAA | 64 65 AAVS1_acc (Bxbl) targeting — 3' junction GTGAGTTTGCCAAGCAGTCA

TTG AAVS1_acc (C31) targeting — 5’ junction CCGGAACTCTGCCCTCTAA | 66 CCGTCCTCGAAGTTCATCAC CCCACAACGAGGACTACACC | 68 AAVS1 ace (C31) targeting — 3’ junction GTGAGTTTGCCAAGCAGTCA

ATC CAAGATCCGCCACAACATCG | 70 CGATGCCTGCTTGCCGAATAT | 71 Bxb1 Donor vector integration {attR) — AAVS1

AGGAC CATG TTTTGGAGTACGTCGTCTTTA | 72 CTGGCAACTAGAAGGCACAG | 73 Bxb1 Donor vector integration (attL) — AAVS1

GG TCG CAAGATCCGCCACAACATCG | 74 CTGGCAACTAGAAGGCACAG | 75 Bxb1 Donor vector integration (attP) — AAVS1

AGGAC TCG CATGGTGAGCAAGGGCGAG | 76 AGTCACGACGTTGTAAAACG | 77 C31 Donor vector integration {attR) — AAVS1

GAG AC TTTTGGAGTACGTCGTCTTTA | 78 GTGAAATTTGTGATGCTATTG | 79 C31 Donor vector integration ({attL) — AAVS1

GG C CATGGTGAGCAAGGGCGAG | 80 81 C31 Donor vector integration {attP) — AAVSI CGGACCACACCGGCGAAGTC

GAG GTGCTGCTGCCCGACAACCA | 82 GAACCTGCGTGCAATECATET | 83 BAC vector integration {(attR) — AAVS1

CTAC T TTTTGGAGTACGTCGTETTTA | 84 85 BAC vector integration {attl) — AAVSI GTGAGTTTGCCAAGCAGTCA

GG Integration integrity — 2-15 kb payload CTCGGCGCGCCATAACTTCG | 86 GTGAGTTTGCCAAGCAGTCA

88 CGATGCCTGCTTGCCGAATAT | 89 loxP/FRT cassette excision — AAVSI CCGGAACTCTGCCCTCTAA

CATG GCTGCAAGAACTCTTCCTCAC | 90 91 lox251/F3 cassette excision — AAVS1 6 GTGAGTTTGCCAAGCAGTCA Optogenetic loxP cassette excision —ASAP2f | TTCGGGTCACCTCTCACTCC | 92 CTCACAGTCGTCTCCATGGT Optogenetic lox251 cassette excision — CAACATTCTGGGGCATAAAC | 94 95

GGCTCCATCGTAAGCAAACC ASAP2f TGG Optogenetic loxP cassette excision — 96 97

TTCGGGTCACCTCTCACTCC TTACGTCGCGATGAGTCGAC jRCaMP1b Optogenetic lox251 cassette excision — GGTCAGGTAAACTACGAAGA | 98

GGCTCCATCGTAAGCAAACC jRCaMP1b G Optogenetic loxP cassette excision — 100 TTCGCAATTCGAATGAGAGG 101

TTCGGGTCACCTCTCACTCC miRFP703 C Optogenetic lox251 cassette excision — 102 103

GCTTCATCCGTTTCGAGCTG GGCTCCATCGTAAGCAAACC miRFP703 Optogenetic loxP cassette excision — AIMA TTCGGGTCACCTCTCACTCC | 104 CTCACAGTCGTCTCCATGGT CAACATTCTGGGGCATAAAC | 106 107 Optogenetic 10x251 cassette excision — AIMA 166 GGCTCCATCGTAAGCAAACC CAACATTCTGGGGCATAAAC | 108 109 integration integrity — ASAP2f-jRCaMP1b TTACGTCGCGATGAGTCGAC

TGG GGTCAGGTAAACTACGAAGA | 110 TTCGCAATTCGAATGAGAGG 111 integration integrity — jRCaMP1b-miRFP703

G C Integration integrity — miRFP703-ASAP2f GCTTCATCCGTTTCGAGCTG | 112 CTCACAGTCGTCTCCATGGT 114 CCATGATATAGACGTTGTGGC | 115 KCNH27/Acc targeting — 5’ junction ACCCGGTCCTTGGTGTTTTC

TGTTG CGGCAGTTGGGATTCGTGAA | 116 117 KCNH2+7Ac targeting — 3’ junction ATTTCCAATCCTCAGGGGCG

TTG 118 CGATGCCTGCTTGCCGAATAT | 119 Donor vector integration (attR) — KCNH2 ACCCGGTCCTTGGTGTTTTC CATG

A GCTGCAAGAACTCTTCCTCAC | 120 121 Donor vector integration (attL} — KCNH2 ATTTCCAATCCTCAGGGGCG

G 122 CGATGCCTGCTTGCCGAATAT | 123 foxP cassette excision — KCNH2 ACCEGGTCCTTGGTGTTTTC

CATG GCTGCAAGAACTCTTCCTCAC | 124 125 fox251 cassette excision — KCNH2 6 ATTTCCAATCCTCAGGGGCG CAAGGAGGCAGGTGGTGTA | 126 127 Screen KCNH2 exon 7 variants CCTCCAACTTGGGTTCCTCC

G integration of reporter construct into NR2F2~ | GCGGACCACTTTCATGCTGA | 200 201 junction TTC CTCCTCGCCCTTGCTCACCAT integration of reporter construct into NR2F2 — | CGGCAGTTGGGATTCGTGAA | 202 CAGGATGTTAATCCACGGAG 203 3’ junction TTG GGTC

1. Cas9 RNP & sgRNA synthesis Cas9 protein was either purchased (IDT) or kindly provided by N. Geijsen (D’Astolfo et al. 2015). Candidate gRNAs with high specificity were identified around the intended mutation site using the bioinformatics tool, CRISPOR (Concordet & Haeussler 2018}, or were previously published (Wang et al. 2015). The gRNAs were synthesised as chimeric single gRNAs (sgRNAs) by in vitro transcription (Branddo et al. 2021).

2. hiPSC culture, clonal isolation and transfections hiPSCs were maintained in StemFlex™ Medium on laminin-521 (LN521; BioLamina)-coated (1.5 ug/cm?) plates. Cells were passaged by dissociating with either 1x TrypLE Select or Accutase® solution (Sigma). For subcloning, dissociated hiPSCs were filtered to remove cell aggregates before being clonally isolated using the single-cell deposition function of a BD FACSAria™ {li (BD Biosciences). Here, single hiPSCs were deposited directly into each well of a LN521-coated (1.8 ug/cm?} 96-well plate. To assist with clonal recovery, the culture media contained RevitaCell™ Supplement (1:100) for 72h as well as the anti- microbial Primocin (invivoGen) for 7 days. Media was changed every 3 days for 2 weeks, after which the cells were replicated for screening and archiving (Brandäo et al. 2021). intracellular delivery of DNA, RNA or protein into the hiPSC line, LUMCO020iCTRL-06 (Zhang et al. 2014) or a subclone of the line, was accomplished by either electroporation or lipofection using conditions previously described (Brandäo et al. 2021) . Electroporation was used for delivery of Cas9-gRNA RNP complexes along with targeting constructs or for the delivery of the BAC _attB(bxb) vector, using protocol #6 (1100 V, 30 ms, 1 pulse) of the Neon Transfection System (ThermoFisher). All other transfections were performed using Lipofectamine™ Stem Transfection Reagent. Prior to transfecting, the medium on the hiPSCs (at “30% confluency), was changed to Opti-MEM™ | Reduced Serum Medium with RevitaCell™ Supplement {1:100). 4 h after transfection, an equal volume of StemFlex was added to the hiPSCs, and the medium completely refreshed 24 h later.

3. Genomic DNA (gDNA) extraction For hiPSCs cultured in 96 well-plates, gDNA was extracted using QuickExtract™ (Lucigen). Cells were resuspended in 30 pl QuickExtract and incubated at 65°C for 15 min, followed by 68°C for 15 min and 98°C for 10 min using a thermocycler. For hiPSCs cultured in other formats, gDNA was extracted using the High Pure PCR Template Preparation Kit (Roche} according to the manufacturer's instructions.

4. Golden Gate (GG) reaction Components amplified by PCR from DNA vectors were treated with Dpnl (NEB) before purifying the PCR product using the Wizard® SV Gel and PCR Clean-Up System kit (Promega). All oligonucleotides were 5’ phosphorylated. Components that were used in multiple constructs (e.g. pCAG and pA signal) were also cloned into a pSMART vector backbone using the CloneSmart® HCKan Cloning Kit (Lucigen). The GG assembly reactions were performed in a total volume of 15 ul and included 80 fmol of each component, 40 fmol destination vector, 5 U restriction enzyme, 200 U T4 DNA Ligase (NEB) in ligation buffer with 100 ug/ml bovine serum albumin (BSA). The reaction was performed in a thermocycler using the program: 37°C for 3 min then 16°C for 4 min (30 cycles); followed by steps of 37°C, 50°C and 80°C, each for 5 min. To remove unligated DNA fragments, the reaction mix was treated with Plasmid-Safe™ ATP-Dependent DNase (Lucigen). Finally, 5 u of the resulting reaction was transformed into the competent E. coli strains indicated.

5. Construction of the Bxb1-LP cassette The components of the Bxb1-LP cassette were first PCR-amplified from other vectors and subsequently ligated together using the NEBuilder® HiFi DNA Assembly Cloning kit (NEB) to generate the resulting vector, pENTR-eGFP-attP(bxb)-*bsdR. Briefly, the fragments amplified comprised of: the backbone of the cloning vector pENTR/D-TOPO; a loxP sequence together with a PGK promoter; an eGFP reporter; and a blasticidin resistance gene lacking an initiation codon (*bsdR). The primers included overlap sequences between adjacent fragments and were also used to introduce the /oxP257 and Bxb1-specific attP sequences into the final vector.

6. Construction of the PhiC31-LP cassette The components of the PhiC31-LP cassette were first PCR-amplified from other vectors and subsequently ligated together using the NEBuilder® HiFi DNA Assembly Cloning kit (NEB) to generate the resulting vector, pENTR-mCherry-attP(PhiC31)-*BleoR. Briefly, the fragments amplified comprised of: the backbone of the cloning vector pENTR/D-TOPO; a FRT sequence together with a PGK promoter; an mCherry reporter; and a bleomycin resistance gene lacking an initiation codon (*bleoR). The primers included overlap sequences between adjacent fragments and were also used to introduce the F3 and PhiC31-specific attP sequences into the final vector.

7. Generation of the AAVS1-Bxb1 and AAVS1-acc hiPSC line To target the Bxb1-LP cassette to the adeno-associated virus integration site (AAVS1) within intron 1 of PPP1R12C, the targeting vector, AAVS1-Bxb1-LP-TC, was generated by: the cassette was PCR-amplified with primers containing overlap sequences to clone via NEBuilder® HiFi DNA Assembly into AAVS1 SA 2A Neo CAG_RTTA3 (Sim et al. 2016) (Addgene, #60431) digested with Hindil! (NEB). The resulting targeting vector (AAVS1-Bxb1-LP-TC) therefore had the Bxb1-LP cassette flanked by ~800 bp homology arms. AAVS1-Bxb1-LP-TC along with Cas9-AAVS1 gRNA RNP complex (gRNA: 5/’- GGGGCCACTAGGGACAGGAT-3'(SEQ ID NO: 5) } were electroporated into LUMC0020iCTRL-06 hiPSCs. Following recovery and expansion of the electroporated cells, eGFP* hiPSCs were clonally isolated. Targeted clones were identified by PCR screening over the 5’ and 3’ homology arms. The resulting AAVS1-acc hiPSC clone selected contained a single integration event of the Bxb1-LP cassette as determined by ddPCR CNV assays for eGFP and BsdR.

8. Generation of the AAVS1-PhiC31 hiPSC line To target the PhiC31-LP cassette to the adeno-associated virus integration site (AAVS1) within intron 1 of PPP1R12C, the targeting vector, AAVS1-PhiC31-LP-TC, was generated by: the cassette was PCR- amplified with primers containing overlap sequences to clone via NEBuilder® HiFi DNA Assembly into AAVS1 SA _2A_Neo_CAG_ RTTA3 (Sim et al. 2016) (Addgene, #60431) digested with Hindi (NEB). The resulting targeting vector (AAVS1-PhiC31-LP-TC} therefore had the PhiC31-LP cassette flanked by ~800 bp homology arms. AAVS1- PhiC31-LP-TC along with Cas9-AAVS1 gRNA RNP complex (gRNA: 5'- GGGGCCACTAGGGACAGGAT-3' ( SEQ ID NO:5)) were electroporated into LUMCOQ20iCTRL-06 hiPSCs. Following recovery and expansion of the electroporated cells, mCherry” hiPSCs were clonally isolated. Targeted clones were identified by PCR screening over the 5’ and 3’ homology arms. The resulting AAVS1-PhiC31 hiPSC clone selected contained a single integration event of the PhiC31-LP cassette as determined by ddPCR CNV assays for mCherry and BleoR.

9. Construction of Bxb1 donor cloning vectors Three Bxb1 donor vectors were constructed for the cloning of various DNA payloads (Figure 9a). For inserting DNA payloads <20 kb, the pBR_attB{bxb}_lox cloning vector was used. This vector was built by digesting the plasmid, pEFBOS_crelRESBsd, with EcoRI (NEB) and ligating with a gBlock that contained a Bxb1-specific attB site, as well as loxP and loxP257 sequences. The cloning vector, p15_attB(bxb)_lox,

was constructed for the cloning of 20 — 50 kb DNA payloads. A DNA sequence containing an ampicillin resistance cassette as well as the p15 origin of replication was PCR-amplified and inserted into the pBR_attB(bxb}_lox donor vector that had been linearised with Pvul and Nsil (both NEB) using NEBuilder® HiFi DNA Assembly.

Lastly the cloning vector, pBR_attB(bxb)_ccdB_lox, was developed for modular construction of multi- component synthetic circuits. In the first step, 3 DNA fragments (EF1a promoter plus Bxb1-attB and loxP sequences; b-lactamase (lacZ) cassette; /oxP257 sequence} were PCR-amplified using primers that incorporated the recognition site for Bpil and specific 4 nucleotide (nt) overhangs to ensure the correct orientation and sequence of fragments in the final construct. In addition, the primers amplifying the lacZ cassette included the recognition site for Esp3l and a further two unique 4 nt overhangs. The pAGM4673 plasmid (Weber et al. 2011) (Addgene #48014) and the 3 PCR products were assembled as a GG reaction using Bpil (ThermoFisher), and transformed into competent DH10p £. coli (NEB). For the second step, a ccdB counterselectable marker was PCR-amplified with primers containing the recognition site for Esp3l and 4 nt overhangs to enable the replacement of the lacZ cassette by GG assembly, thereby generating the pBR_attB(bxb)_ccdB_lox donor vector.

10. Construction of PhiC31 donor cloning vector The pBR_attB(PhiC31} FRT cloning vector was built by a two-step digestion-ligation cloning method. Firstly, the plasmid pEFBOS_crelRESBsd was digested with EcoR! (NEB) and ligated with a gBlock sequence that contained a PhiC31-specific attB site, a BamHI site, and a FRT-F3 sequence. Subsequently, this resulting plasmid was digested with BamHI (NEB) and ligated with a gBlock that contained a FRT sequence.

11. Construction of vectors for evaluating integration of different DNA payload sizes The insertion of DNA payloads between ~2 — 50 kb into either the pBR_attB(bxb}_lox or p15_attB(bxb}_ lox donor vectors was performed by subcloning fragments from a bacterial artificial chromosome (BAC) carrying the human KCNH2 gene (RP11-10L20) via recombineering (Figure 9C} (Fu et al. 2010). To modify RP11-10L20 to enable the targeted integration of the complete BAC construct (173 kb} into the AAVS1-acc hiPSCs, recombineering was used to replace the loxP sequence in the BAC with a DNA fragment including the EF1a promoter, Bxb1-attB and a kanamycin-resistance cassette that was PCR-amplified from a R6K-vector containing this insert.

12. Integration of donor vectors into the AAVS1-acc hiPSCs

Unless stated, 1.2 ug of the donor vectors along with 0.8 ug of the plasmid, pCAG-NLS-HA-Bxb1 (Hermann et al. 2014) (Addgene #51271), were transfected by lipofection into the AAVS1-acc hiPSCs. To integrate the modified RP11-10L20 BAC construct into the AAVS1-acc hiPSCs, BAC_attB(bxb} was co- electroporated with pCAG-NLS-HA-Bxb1. For both approaches, ~3 days post transfection the cells were harvested and passaged so that they were ~5% confluent the following day. To enrich for integrated hiPSCs, blasticidin (2 ug/ml, Sigma) was added to the culture medium for a period of 12 days. Similarly,

1.2 ug of the pBR_attB(phiC31)_FRT plasmid along with 0.8 ug of the plasmid, pBCW (Ohtsuka et al. 2015) (Addgene # 62658), were transfected by lipofection into the AAVS1-acc hiPSCs. ~3 days post transfection the cells were harvested and passaged so that they were “5% confluent the following day. To enrich for integrated hiPSCs, zeocin (15 pg/ml, Sigma) was added to the culture medium for a period of 6 days. In both cases, donor vector integration was confirmed via a PCR screening strategy to detect the formation of the two new recombination sites, attR and attl.

13. Excision of auxiliary sequences Cells were transfected by lipofection with either a Cre recombinase-expressing plasmid (1.6 ug, pEFBOS_CrelRESpuro (Davis et al. 2008)), StemMACS™ Cre Recombinase mRNA (200 ng, Miltenyi Biotec), a Flpo recombinase -expressing plasmid (1.6 ug, pCAGGs-Flpo-IRES-puro (Kranz et al. 2010), or a plasmid expressing both Cre and Flpo recombinase (1.6 pg, pEfBOS_flpOQ_2A_crelRESPuro (SEQ ID NO 198}). For hiPSCs transfected with the plasmid, selection with puromycin (1 pg/ml, Sigma) was initiated 24 h post-transfection and maintained for 48 h. Genotyping PCR was used to confirm the loxP-, loxP257- , FRT- or FRT-F3-flanked sequences were excised. 14, Droplet digital PCR (ddPCR) ddPCR was performed and analysed using a thermocycler, the Q200 AutoDG and QX200™ Droplet Digital PCR System, and QuantaSoft software (all Bio-Rad). Assays comprising of premixtures of a forward and reverse primer {18 uM each) with a FAM- or HEX-conjugated hydrolysis probe (5 pM) were either purchased from Bio-Rad, based on previous publications (Roberts et al. 2017), or designed based on pre-defined criteria (Bell et al. 2018). Details regarding the assays are listed in Table 3. Reactions (22 ul} were prepared with 2x Supermix for probes with no dUTP (Bio-Rad), 900 nM of each primer, 250 nM of each probe, and 30-100 ng of gDNA digested with 2-5 U of Hindlll, Haelll or Msel (NEB) depending on the sequence of the amplicon. Droplet generation, PCR amplification and analysis were all performed according to the manufacturer's instructions. For copy number assays, the two-copy autosomal gene RPP30 gene was used as a reference. Table 3. Primer-Probe sets used for ddPCR Spee pe Ea Copy Reverse GGGTGTTCTGCTGGTAG | 129 Roberts et gf. Mol AGATCCGCCACAACATC | 130 Forward TGGCAACCTGACTTGTA | 131 em oe BsdR This study number Primer CTATC CGACAGGTGCTTCTCGA | 133 Forward GTGAGCAAGGGCGAGG | 134 ee number Primer GGC TCAAGGTGCACATGGA 136 Forward AGTTGACCAGTGCCGTT | 137

J BleoR This study number Primer AAG AGCCGGTCGGTCCAGA | 139 Forward GCATTCTAGTTGTGGTT | 140 Ee Bxbl-gttP | Integration This study Primer TTGAG CGTGGTTTGTCTGGTCA | 142

KCNH2_F627L | CT+T +GG+G C+AA CGT | 163 Probe C FAM-ZEN-IBFQ KCNH2_G628 | CTT +T+A+G CA+A 164 KCNH2_N633 | TCC C+A+G +CAC CA+A 165 RE.

Co ros | EE EEE Copy dHsaCNS6525 | hg19{chr7:150641909- Copy dHsaCP10001 | hg19|chr7:150695400- Copy dHsaCNS7915 | hg19jchr7:150549559- TMEM176 | Copy dHsaCNS9381 | hgl9ichr7:150488415- Copy dHsaCNS4295 | hg19}chr7:150725518- + symbol indicates the following nucleotide is a locked nucleic acid attP:attR assay.

To determine the recombination efficiency of the donor plasmids into the Bxb1-LP or PhiC31-LP, probes were designed to detect the attP and/or the attR sites in the transfected hiPSCs.

For both events the forward primer for that specific LP was common, while the reverse primers were specific for either the non-integrated or integrated locus.

For amplicons amplified from the non- integrated population only one of the probes annealed, while for amplicons obtained from the integrated population both probes hybridized resulting in a stronger fluorescent signal that made it possible to distinguish droplets from each target.

To optimise amplification conditions and confirm the specificity of the assay, gBlocks matching the two expected amplicons for either Bxb1- or PhiC31- mediated integration were mixed in differing ratios and used as template DNA, with a strong correlation (R2=0.9993 for Bxb1; R?=0.9989 for PhiC31) seen between the expected and observed frequencies for both assays (results not shown). KCNH2 variants. individual probes were designed for each of the 12 missense mutations.

To improve target specificity, 2-5 locked nucleic acids were included per probe. Amplification conditions for each probe were optimised for discriminating amplicons containing that specific missense mutation. Droplets were analysed using the “absolute quantification” option.

15. Generation of optogenetic reporter hiPSC lines The assembly of the individual and multi-parameter reporter donor vectors was based on the modular and hierarchal cloning system, MoClo (Weber et al. 2011). Briefly, the required components for the expression of these reporters (i.e. promoter sequence, localisation signal, coding sequence and polyA signal) were PCR-amplified using primers that incorporated the recognition site for the type IIS enzyme Bsal and previously designated overhang sequences for positioning and orientation of the components (Weber et al. 2011; Andreou & Nakayama 2018). The only exception was the CDS for jRCaMP1b, which was synthetic designed so as to introduce silent mutations to destroy Bpil and Bsal recognition sites present in the original CDS (Dana et al. 2016). The components for each of the individual optogenetic sensors were first assembled in Level 1 destination vectors included in the MoClo Toolkit (Addgene #1000000044) by GG assembly with the restriction enzyme, FastDigest Eco31l (ThermoFisher), thereby generating the intermediate Transcriptional Unit (TU) vectors. The reporter construct for ASAP2f was assembled into 2 different Level 1 destination vectors, while the constructs for jRCaMP1b and miRFP703 were each assembled into Level 1 destination vectors for positions 2 and 3 respectively. The resulting TU vectors were subsequently assembled either individually or in combination into the donor vector pBR_attB{bxb)_ccdB_lox by digestion with Bpil. Dummy and end-linker vectors from the MoClo Toolkit were included as required in the GG assembly reaction. The ensuing donor vectors (pBR- attB_ASAP2f; pBR-attB_jRCaMP1b; pBR-attB_Lck-miRFP703; pBR-attB_AIMA_donor) were transformed into Stbi2™ E. coli for ccdB counterselection. Each donor vector was separately integrated into the AAVSI-acc hiPSCs, followed by excision of the auxiliary sequences. Clonal hiPSC lines were derived for the single reporter targeted cells, while enrichment for the AAVS1-AJMA hiPSCs was performed by flow cytometric sorting of cells co-expressing the 3 reporters. Genotyping PCRs confirmed targeted integration, as well as no rearrangement within the TUs of the AAVS1-AJMA hiPSCs.

16. Generation of KCNH2*/AC hiPSC line

The vector to target the Bxb1-LP cassette to the KCNH2 locus (KCNH2-Bxb1-LP-TC) was generated by PCR-amplifying the Bxb1-LP cassette with primers containing 80 bp overhangs complementary to endogenous sequences ~8.5 kb (5°) and ~8.7 kb (3') of KCNH2. The resulting PCR product was cloned into pMini T2.0 (NEB).

KCNH2-Bxb1-LP-TC along with a Cas9-KCNH2 gRNA RNP complex (RNA: 5'-CTGGTTGTGCTGACTGTGCT- 3’ {SEQ ID NO: 166) } was then electroporated into a subclone of LUMCOOZOICTRL-06 in which one copy of KCNH2 had previously been deleted. Following recovery and expansion of the electroporated cells, eGFP’ hiPSCs were clonally isolated. Clones in which the Bxb1-LP cassette were targeted to the deleted allele were identified by PCR screening over the 5’ and 3’ homology arms. The resulting KCNH27/AC hiPSC line selected contained a single integration event of the Bxb1-LP cassette as determined by ddPCR CNV assays for eGFP and BsdR, as well as one copy of KCNHZ.

17. Construction of KCNH2 wildtype and variant donor vectors The construction of the donor vectors containing the various KCNH2 genomic sequences were accomplished based on recombineering strategies previously described (Wang et al. 2014; Fu et al.

2010). Briefly, to seamlessly introduce the variants into KCNH2, a counterselection cassette (ccdB-Amp) was first introduced to replace exon 7 in the BAC, RP11-10L20. Next, synthetic double-stranded DNA fragments that introduced specific missense mutations in exon 7 of KCNH2 (Table 4) were pooled and amplified by PCR, before being electroporated into the bacteria to replace the counterselection cassette. Colonies that subsequently grew in the absence of L-arabinose were then screened by PCR and Sanger sequencing to identify recombined BACs for each of the 12 mutations.

Table 4. KCNH2 variants introduced in KCNH2/A< hiPSCs, annotated and with corresponding gBlock sequence SNP | Nucle | Protein | Interpretat | Assorted | gBlock | SIO Comments 12516 | G>C syndrome ccttccctgtcccccagctgatcgggctgctgaa mutation gactgegeggcetgetgeggetggigegestgec introduced geggaagetggategetacicagagtacggeg into cggccgtgctgttcttgctcatgtgcaccttCCc adjacent cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctgggc gaccagataggcaaaccctacaacagcagcg gectgggcggcccctccatcaaggacaagtat gtgacggcgcictacttcaccttcagcagcctc accagtgigggcticggcaacgtctctcccaac accaactcagagaagatcttciccatctgcgtc atgctcattggctgtgagtgtgcccaggggcgg BCEgCgEggagagcccacggiggaggaaccC aagttggaggaaactgaggctgctagccgggc ca rs1994 | c.1681 | A561T pathogenic Long QT cttgccceccttgccccatcaacggaatgtgcc | 168

72921 | G>A syndrome ccttccctgtcccccagctgatcgggctgcigaa gactgcgcggctgctgcggctggtgcgegtgg gcggaagctggatcgctactcagagtacggcg cggccgtgcigttcttgeicatgtgcaccttigc gctcatcAcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctggge gaccagataggcaaaccctacaacagcagcg gcctgggcggcccctccatcaaggacaagtat gtgacggcgcictacttcaccticagcagcctc accagtgtgggcttcggcaacgtctctcccaac accaactcagagaagatctictccatctgegtc atgctcattggctgtgagtgtgcccagggEceg geggegrggagageccacggtggaggaacce aagttggaggaaactgaggcigctagccgggc ca rs7947 | c.1733 | H578P uncertain cttgccccccttgccccatcaacggaatgigcc | 169

28376 | A>C significance ccttccctgtcccccagctgatcgggcigctgaa gactgegeggetgetgeggetggtgegegtege gcggaagetggategetactcagagtacggeg cggeegtgetgtictigeteatgtgeacctitge gcteategegeaciggetagectgeatetggta cgccatcggcaacatggagcagccacCcatgg actcacgcatcggciggcigcacaacctgggc gaccagataggcaaaccctacaacagcagcg gcctgggcggcccciccatcaaggacaagtat gigacggcgctctacttcaccttcagcagccte accagtgtgggcttcggcaacgtctctcccaac accaactcagagaagatcttctccatctgegtc atgctcattggctgigagtgigcccaggggcgE gegecggggagageccacggtiggaggaacce aagttggaggaaactgaggctgctagcCgggc ca rs1994 | c.1750 | G584S likely Long QT cttgccccccttgccccatcaacggaatgtgcc | 170 73428 | G>A pathogenic; syndrome ccttccctgtcccccagctgatcgggctgctgaa pathogenic gactgegeggetgetgeggetggtgegegtggce gcggaagctggatcgctactcagagtacggcg cggccgtgctgttcttgctcatgtgcacctttgc gctcatcgcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcAgctggctgcacaacctgggc gaccagataggcaaaccctacaacagcagcg gcctgggcggcccctccatcaaggacaagtat gtgacggcgctctacttcaccttcagcagcctc accagtgtgggcticggcaacgtctctcccaac accaactcagagaagatcttctccatctgcgtc atgctcattggctgtgagtgigcccaggggcgE geggeggggagageecacggtggaggaacce aagttggaggaaactgaggctgctagccgggc ca rs1994 | c.1762 | N588D not provided | Long QT cttgccccccttgccccatcaacggaatgtgcc | 171 73431 | A>G syndrome ccttccctgtcccccagctgatcgggctgctgaa gactgegeggctgetgeggetggigegegigge gcggaagctggatcgctactcagagtacggcg cggccgtgctgttcttgctcatgtgcacctttge gctcategcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacGacctgggc gaccagataggcaaaccctacaacageageg gectgggcggcccctccatcaaggacaagtat gîgacggcgctctacttcaccttcagcagcetc accagtgtgggcttcggcaacgtctctcccaac accaactcagagaagatcttctccatctgcgtc atgctcattggcigtgagtgtgcccaggggcgg ECggCgEEgagagcccacggiggaggaaccc aagttggaggaaactgaggctgctagccgggc ca rs1048 | c.1764 | N588K pathogenic Short QT cttgccccccttgccccatcaacggaatgtgcc | 172

94021 | C&G syndrome ccttccctgtcccccagctgatcgggctgctgaa gactgegeggetgetgeggetggtgegegtggce gcggaagctggatcgctactcagagtacggcg cggccgtgctgttcttgctcatgtgcacctttgc gctcategcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaaGctgggc gaccagataggcaaaccctacaacageageg gcctgggcggcccctccatcaaggacaagtat gtgacggegctctacttcaccttcagcagcctc accagtgtgggcttcggcaacgtctctcccaac accaactcagagaagatcttctccatctgcgtc atgctcattggcigtgagigtgcccaggggcgg ECggCgEEgagagcccacggiggaggaaccc aagttggaggaaactgaggctgctagccgggc ca rs1994 | c.1841 | A614V pathogenic Long QT cttgccccccttgccccatcaacggaatgtgcc 173

72944 | C>T syndrome ccttccctgtcccccagctgatcgggctgctgaa gactgegcggctgctgcggctggtgcgcgiggc gcggaagctggatcgctactcagagtacggcg cggccgtgctgttcttgctcatgtgcacctttge gctcatcgcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctgggc gaccagataggcaaaccctacaacagcagcg gectgggcggcccctccatcaaggacaagtat gtgacggTgctctacttcaccttcagcagcctc accagtgigggcticggcaacgtctctcccaac accaactcagagaagatcttciccatctgcgtc atgctcattggcigtgagtgtgcccaggggcgg BCEgCgEggagagcccacggiggaggaaccC aagttggaggaaactgaggctgctagccgggc ca rs1994 | c.1853 | T618! not provided | Short QT cttgccccccttgccccatcaacggaatgtgcc | 174

72947 | C>T syndrome ccttccctgtcccccagctgatcgggctgcigaa gactgegeggetgetgeggetggtgegegtgge gcggaagctggatcgctactcagagtacggcg cggccgtgcigttcttgeicatgtgcaccttigc gctcatcgcgcactggctagcctgcatciggia cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctgggc gaccagataggcaaaccctacaacagcagcg gcctgggcggcccctccatcaaggacaagtat gigacggcgctctacttcaTcttcagcagcctc accagtgtgggcttcggcaacgtetctcccaac accaactcagagaagatcttctccatctgegtc atgctcattggctgtgagtgtgcccagggeceg geggeggggagageccacggtggaggaacce aagttggaggaaactgaggcigctagccgggc ca rs1994 | c.1853 | T618S not provided | Long QT cttgccccccttgccccatcaacggaatgtgcc 175

72947 | C>G syndrome ccttecctgtcccccagctgatcgggcigctgaa gactgegeggcetgetgeggetggtgegegtege gcggaagctggatcgctactcagagtacggeg cggeegtgetgtictigeteatgtgeacctitge gctcatcgcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcggciggcigcacaacctgggc gaccagataggcaaaccctacaaCagcagcg gcctgggcggcccciccatcaaggacaagtat gigacggcgctctacttcaGcttcagcagccte accagtgtgggcttcggcaacgtctcicccaaC accaactcagagaagatcttctccatctgegtc atgctcattggetgtgagtgtgeccaggggesg geggeggggagageccacggtggaggaacce aagiiggaggaaactgaggetgetageeggge ca rs1994 | c.1881 | F627L pathogenic Long QT cttgccccccttgccccatcaacggaatgtgcc | 176

73039 | C>A/G syndrome ccttccctgtcccccagctgatcgggctgctgaa gaCIgCgCBECtgeIgCBECIgEIgCBCgIgEC gcegaagciggatcgctactcagagtacggcg cggccgtgctgttcttgctcatgtgcacctttgc gctcatcgcgcactggctagcctgcatctggta cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctgggc gaccagataggcaaaccctacaacagcagcg gcctgggcggcccctccatcaaggacaagtat gtgacggcgctctacttcaccttcagcagcctc accagtgtgggcttGggcaacgtctctcccaac accaactcagagaagatcttctceatetgegte atgctcattggcigtgagigtgcccaggggcgg ECggCgEEgagagcccacggiggaggaaccc aagttggaggaaactgaggctgctagccgggc ca rs1219 | c.1882 | G6285 pathogenic Long QT cttgccccccttgccccatcaacggaatgtgcc | 177 silent

12507 | G>A syndrome ccttccctgtcccccagctgatcgggctgctgaa mutation gactgegcggctgctgcggctggtgcgcgiggc introduced gcggaagctggatcgctactcagagtacggcg into cggccgtgctgttcttgctcatgtgcacctttge adjacent gctcatcgcgcactggctagcctgcatctggta amino acid cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctgggc gaccagataggcaaaccctacaacagcagcg gectgggcggcccctccatcaaggacaagtat gîgacggcgctctacttcaccttcagcagcetc accagtgtgggcttTAgcaacgtctctcccaac accaactcagagaagatcttciccatctgcgtc atgctcattggcigtgagtgtgcccaggggcgg BCEgCgEggagagcccacggiggaggaaccC aagttggaggaaactgaggctgctagccgggc ca rs1994 | c.1898 | N633S pathogenic Long QT cttgccceecttgccccatcaacggaatgtgcc | 178

72961 | A>G syndrome ccttccctgtcccccagctgatcgggctgcigaa gactgegeggetgetgeggetggtgegegtgge gcggaagctggatcgctactcagagtacggcg cggccgtgcigttcttgeicatgtgcaccttigc gctcatcgcgcactggctagcctgcatciggia cgccatcggcaacatggagcagccacacatgg actcacgcatcggctggctgcacaacctggge gaccagataggcaaaccctacaacagcagcg gcctgggcggcccctccatcaaggacaagtat gtgacggcgcictacttcaccticagcagcctc accagtgtgggcttcggcaacgtcictcccaGc accaactcagagaagatcttctccatctgcgtc atgctcattggctgtgagtgtgcccagggEceg BCEECgEgEgagagcccacggiggaggaaccc aagttggaggaaactgaggctgctagccgggc ca introduced mutations are indicated in capita! letters.

The sequence in the BAC corresponding to the KCNH2 genomic region deleted in the KCNH2*A< hiPSCs was subsequently subcloned from both wildtype RP11-10L20 as well as clones carrying the introduced variants into p15_attB(bxb}_lox by recombineering. The resulting colonies were screened by PCR to confirm subcloning and the plasmids retransformed to ensure the resulting KCNH2 donor vectors were pure. Sanger sequencing also confirmed the presence of each missense mutation, and that the attB and lox sequences were correct. Finally, the integrity of each of the donor vectors was evaluated by restriction analysis. Bacterial cultures of the KCNH2 variant donor vectors were pooled and a 200 mi culture grown overnight at 30°C, while the KCNH2 wildtype donor vector (p15-att8_KCNH2_wt_donor) was cultured separately. Plasmid DNA was purified using the NucleoBond® Xtra Midi Kit (Macherey Nagel) following manufacturer's instructions.

18. Generation of KCNH27ACWt and KCNH27/Accvariant hipSe lines For the generation of the KCNH2*A°W line, a similar procedure to that used to generate AAVS1-reporter hiPSCs was performed, except that 3.2 ug of p15-attB_KCNH2_wt_donor was transfected into the KCNH27AC hipSCs. Cells were selected with blasticidin for 6 days, followed by transfection of pEFBOS_ CrelRESpuro and selection with puromycin. The resulting KCNH2*7/A°W hipSCs were clonally isolated by single-cell deposition and identified by genotyping PCR. To multiplex the generation of the KCNH22« vt nes, 0.6 ug of the pooled 12 KCNH2 variant donor vectors was transfected because of toxicity observed when transfecting higher amounts of plasmid DNA. The blasticidin selection strategy was also modified, with the cells maintained in culture medium containing 2 pg/ml blasticidin over 3 passages (14 days). Excision of the auxiliary sequences was performed by two rounds of Cre recombinase mRNA transfection. Following single-cell deposition, genotyping PCR detected clones that had undergone both integration and excision steps, while Sanger sequencing identified the heterozygous KCNH2 variant that was introduced.

19. Differentiation and culture of hiPSC-derived cardiomyocytes (hiPSC-CMs) Monolayer-based differentiation of the hiPSCs to cardiomyocytes using small molecules, and the subsequent cryopreservation of the hiPSC-CMs at day 21 of differentiation was performed as previously described (Campostrini et al. 2021; van den Brink, Brandâo, et al. 2020) . For replating the hiPSC-CMs, the frozen cells were thawed as described (Campostrini et al. 2021; van den Brink, Brandäo, et al. 2020) and seeded as required in mBEL medium.

20. Flow cytometric analysis

A single cell suspension of hiPSCs or hiPSC-CMs was obtained by dissociating the cells with TrypLE Select and filtering the cell suspension. Cells were fixed and permeabilised using the FIX and PERM™ Cell Permeabilization Kit (Invitrogen) according to manufacturer's instructions. The hiPSC-CMs were incubated with the conjugated antibodies cTnT-Vioblue or cTnT-FITC (1:50, Miltenyi Biotec, #130-120- 402 or #130-119-575). All antibodies were diluted in permeabilization medium (medium B; Invitrogen). The data was acquired using a MacsQuant VYB flow cytometer (Miltenyi Biotec) and analysed using FlowJo software (FlowJo).

21. Fluorescence imaging For imaging the hiPSCs and hiPSC-CMs, the cells were seeded on Falcon® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate (Corning), with images of the fluorescent reporters acquired using either a Leica SP8 microscope equipped with a Dragonfly VR spinning disk (Andor) using a 63x oil objective (NA 1.4), or the EVOS™ M7000 Cell Imaging System (ThermoFisher) using a 40x objective. For visualizing the sarcomeres, the hiPSC-CMs were fixed and permeabilized with the FIX and PERM™ Cell Permeabilization kit, and labelled with an antibody specific for a-actinin (1:250, Sigma-Aldrich #A7811), followed by an Alexa Fluor® 350-conjugated secondary antibody (1:500, ThermoFisher, #A-11045). images were captured using the EVOS™ M7000 Cell Imaging System at 40x magnification.

22. Optical evaluation of the hiPSC-CMs 4 - 5 x 10° hiPSC-CMs differentiated from the optogenetic reporter hiPSCs were seeded per well on 96- well imaging microplates pre-coated with Matrigel (1:100; Corning) in mBEL medium. Medium was refreshed the next day and every 2-3 days thereafter, with analysis performed 7 days after plating. Before performing baseline measurements, wells were refreshed with 200 mL mBEL medium and left for 60 min at 37°C to recover. After baseline measurements, the hiPSC-CMs were refreshed with 100 uL of mBEL + drug compound and incubated for 5 min at 37 °C before recording. For drug evaluation, the compounds used were E4031, verapamil (both Tocris Bioscience) and isoprenaline (Sigma-Aldrich). All drugs were reconstituted in DMSO (Sigma-Aldrich), with solutions prepared to ensure a final concentration of 0.1% DMSO in each well. Vehicle incubations were done similarly using mBEL + 0.1% DMSO. All measurements were made with cells paced at 1.2 Hz using a pair of field stimulation electrodes placed in the culture medium, except for the isoprenaline measurements which were performed on spontaneously beating cells. For measurements, a Leica DMI6000B imaging system (Leica Microsystems} equipped with 470, 565 and 656 nm lasers was used to record the voltage, Ca?’ flux and motion-contraction signals at 40 frames per second. The microscope was fitted with an environmental chamber that allowed for the measurements to be performed at 37°C and 5% CO,. The analyses for the voltage and Ca?’ flux were performed using Image] (NIH) and algorithms developed in-house (van Meer et al. 2019). For the motion-contraction signal, the analyses were performed using the MUSCLEMOTION Software (Sala et al. 2018).

23. Multielectrode array (MEA) recordings of hiPSC-CMs 4 — 6 x 10° hiPSC-CMs differentiated from the KCNH2AWT and KCNH2*/ACCA561T hipSCs (4-5 independent differentiations per line} were seeded per well of a Matrigel-coated 96 well Lumos MEA plates (Axion BioSystems, Inc.) in 5 ul mBEL medium supplemented with RevitaCell™ (1:200), and incubated for 1 h at 37°C, 5% CO; to allow cell attachment. Wells were then supplemented with an additional 150 pl mBEL medium, with medium refreshed the next day and every 2-3 days thereafter. At d11-12 post-thaw, recordings were performed using the Maestro Pro multiwell MEA platform (Axion BioSystems, Inc.). Field potential (FP) recordings of spontaneous beating hiPSC-CMs were performed at least 1 h after a medium change. Prior to the recording, the hiPSC-CMs were equilibrated inside the device for 10 min. Recordings were performed at 37°C, 5% CO: for 3-4 min using Cardiac Standard filters and amplifiers in spontaneous cardiac mode (12.5 kHz sampling frequency; 0.1-2000 Hz band pass filter).

24. Statistical analysis All data is presented as mean, with detailed statistics and statistical significance indicated in the figure legends. The statistical power was determined using the Two-tailed T test for unpaired measurements. Differences were considered statistically significant at P <0.05. Sample sizes are indicated in the figures. Statistical analysis was performed with GraphPad Prism 8 software (v8.2.0, GraphPad). Results

1. Generation of hiPSC acceptor lines for Bxb1- and phiC31-mediated integration in order to validate our strategy for the targeted integration of large genomic fragments into hiPSCs, we first used CRISPR/Cas9-mediated homologous recombination to target our LP constructs to the AAVS1 locus in the well-characterised hiPSC line, LUMCOOZOICTRLS (Figure 1A) (Zhang et al. 2014; Brando et al. 2020). The AAVS1 locus is a hotspot for adeno-associated virus integration and is located in intron 1 of the gene encoding protein phosphatase 1, regulatory subunit 12C (PPP1R12C) on chromosome 19 {Sadelain et al. 2011). It has previously been demonstrated as a site in hiPSCs that favours stable, long-

term expression of transgenes introduced there, including in many differentiated derivatives such as cardiomyocytes (hiPSC-CMs) (Sun et al. 2020). The LP cassettes for both Bxb1 and phiC31 were similarly designed and consisted of a pGK promoter driving expression of a fluorescent protein (eGFP for Bxb1-LP; mCherry for phiC31-LP}, an attP site recognised by the corresponding tyrosine recombinase, and an antibiotic positive selection marker that lacked an ATG initiation codon (blasticidin (bsd) for Bxb1-LP; bleomycin (bleo) for phiC31-LP). These cassettes were also flanked by heterotypic recognition sequences for either Cre or Flp recombinase to enable its excision downstream (lox for Bxb1-LP; frt for phiC31-LP). Finally, the LP cassettes were incorporated into a targeting vector that included ~2kb of the PPP1R12C gene sequence.

We subsequently transfected the hiPSCs by electroporation with Cas9 RNP, a previously reported sgRNA designed to generate a double strand break at the AAVS1 locus (Wang et al. 2015), and either one or both of the LP targeting vectors. Because each LP construct was comprised of unique components, this permitted us to generate hiPSC acceptor lines containing either the Bxb1- or phiC31-LP (AAVS1- Bxb1/AAVS1-acc and AAVS1-pC31 respectively). Additionally, we generated an hiPSC line that was biallelically targeted with both LPs (AAVS1-dual), thereby providing us with a cell line containing an orthogonal pair of target sites that do not cross-react. By including a fluorescent reporter in the LP cassette, we were able to identify hiPSCs that had stably integrated the LP and isolate clonal lines by fluorescence-activated cell sorting (FACS). The clones selected to use as the AAV51-Bxb1 and AAVSI- C31 acceptor hiPSC lines expressed either GFP or mCherry respectively, while the AAVS1-dual acceptor hiPSC line expressed both reporters (Figure 1B).

PCR screening confirmed integration of LP cassettes into the AAVS1 locus for each of the hiPSC acceptor lines (Figure 1C), with Sanger sequencing verifying that the LP had been targeted by homologous recombination. Sequencing also confirmed that the attP target sites along with the recognition sequences for Cre and Flp contained no errors in any of the selected lines, and that the AAVS1-dual hiPSC line was biallelically targeted at the AAVS1 locus with both the bxb1- and phiC31-LPs. Finally, using ddPCR we determined that each of the clones selected had either none or just one copy of the genes encoding eGFP, mCherry, bsdR and bleoR, indicating that each acceptor line had at most a single integration of each LP (Figure 1D).

2. Bxb1 mediates highly efficient targeted integration of constructs into hiPSCs without size restrictions To assess whether we could not only integrate constructs into the LPs in the hiPSC acceptor lines but also efficiently enrich and isolate these targeted clones, we developed donor vectors specifically for both the Bxb1- and phiC31-LPs (Figure 2A). A key feature of our strategy was that the donor vector included an Efla promoter followed by an ATG initiation codon and either the attB target sequence recognised by Bxb1 or phiC31. Correct integration of the construct into the respective LP would place the promoter and start codon upstream and in frame with the antibiotic resistance gene previously targeted in the hiPSC acceptor lines, thereby resulting in expression of the gene and enabling enrichment of correctly integrated clones by either blasticidin (Bxb1-LP) or zeocin (phiC31-LP) selection. Additionally, the donor vector contained either heterotypic lox (Bxb1-donor) or frt (phiC31-donor) sequences flanking the location in the vector where the DNA payload is inserted. We initially co-transfected into the AAVS1-dual hiPSC line these empty donor vectors along with a plasmid expressing either Bxb1 or PhiC31 to evaluate the efficiency of the system as well as assess the integration specificity of both serine recombinases. Even prior to antibiotic selection, integration of the Bxb1-donor vector could be detected by diagnostic PCRs that amplified across the two new recombination sites, attR and attL (Figure 2B). This was not observed with the phiC31-donor until after zeocin selection, intimating phiC31 was less efficient at mediating integration. This was also reflected in the number of colonies obtained following selection with 8 — 10-fold more potential recombinants obtained from cells transfected with the Bxb1-donor and Bxb1 integrase (Figure 2C). The specificity of the integrases was also evident with no colonies obtained following antibiotic selection when we mismatched the integrase with the attB donor vector, indicating no crosstalk between the integrases and their target sites. These results also concur with reports using other mammalian cell lines that have found serine recombinases to be highly specific and also Bxb1 to be more efficient at integrating constructs than PhiC31 (Xu et al. 2013; Jusiak et al. 2018). We therefore decided to focus on further evaluating the capabilities of Bxb1 to perform targeted integrations of large DNA payloads into hiPSCs. However, we did also confirm that PhiC31 mediated on-target integration of constructs and that the resulting integrated vector backbone could be excised by flpO recombinase (Figure 11).

Amongst the Bxb1-mediated recombinant colonies obtained following blasticidin selection, diagnostic PCR also detected the attP target sequence within this population indicating that not all cells had integrated the construct (Figure 2B). We therefore developed a digital droplet PCR (ddPCR) assay to quantify the percentage of recombinant cells following selection.

The ddPCR assay consisted of 3 primers for the amplification of both attP and attR target sequences, as well as a reference probe present in both amplicons and a second probe only present in amplicons obtained from cells that had integrated the donor vector (Figure 2D). Using this ddPCR assay, we determined that integration of the

Bxb1-donor had occurred in ~30% of the AAVS1-Bxb1 hiPSCs following 12 days of blasticidin selection (Figure 2E). To evaluate whether the efficiency of integration in this system was independent of the size of the construct to integrate, we designed a series of donor vectors with DNA payloads varying in size from ~2 to 50 kb.

Following co-transfection of each of these donor constructs with Bxb1 and subsequent blasticidin selection, a similar integration efficiency to that obtained for the empty vector was seen for constructs containing payloads up to 10 kb (Figure 2E & 2F). Interestingly, integrating larger payloads was more efficient with nearly 50% of the resulting pool of cells being recombinants (50 kb: 47.8+4.3%). This also did not change when we adjusted the amount of the different donor vectors transfected to match the number of copies of the 50 kb payload that was being delivered to the hiPSCs.

Performing multiple rounds of blasticidin selection (enrichment) on the passaged transfected cells further increased the percentage of hiPSCs that had integrated the 50 kb payload upto a high level of ~80% after 5 rounds of passaging (Figure 10). This also contrasts with the results found if the integration construct contains also the constitutive promoter and start codon for BsdR, where following blasticidin selection only about 7.5% of the resulting hiPSCs have the construct integrated at the desired locus (Figure 12). This is most likely due to random integration of the construct into the cells or only a partial fragment of the construct being correctly targeted.

We then investigated whether there was an upper limit to the size of the DNA payload that could be targeted into the hiPSCs.

We modified a BAC construct to include the necessary components to insert into the Bxb1-LP and enrich for targeted integrants, resulting in a 173 kb construct (Figure 3A & 3B). While the transfection strategy that we previously used to integrate the donor vectors was not successful, electroporation of the modified BAC vector along with Bxb1 integrase resulted in blasticidin- resistant hiPSCs that were positive for attR and attL recombination sites (Figure 3C), with single cell cloning indicating an efficiency of ~4%. To further validate that targeted integration of 173 kb into the

AAVS1 locus had occurred, we used ddPCR to confirm that one of these hiPSC lines had also acquired an additional third copy of the three genes (KCNH2, NOS3 and AOC1) contained on the BAC donor (Figure 3D). Overall these results indicated that there appeared to be no upper limit to the size of the construct that we could target with Bxb1 integrase and that our strategy managed to preserve the genetic integrity of such large DNA payloads.

3. Cre recombinase-mediated excision of auxiliary sequences following donor vector integration As our strategy employs the use of a single serine recombinase, this results in the entire donor vector being integrated into the corresponding LP. However, it has previously been observed that if the backbone of a construct remains incorporated in the genome, this can lead to silencing of either the transgenes or neighbouring genetic elements (Chen et al. 2004; Pham et al. 1996). Conversely, this effect can be prevented or even reversed if these sequences are subsequently excised from the targeted foci (Riu et al. 2005; Davis et al. 2008). Because the DNA payload in the donor vector was flanked with heterotypic lox sequences, this resulted in both the vector backbone as well as the eGFP expression cassette being “floxed” by /oxP257 and loxP sequences respectively when the construct integrated into the bxb1-LP (Figure 4A).

Transiently expressing Cre recombinase in the enriched pool of recombinant hiPSCs led to the removal of these auxiliary sequences, leaving the integrated DNA payload along with a single copy of loxP257 and /oxP sequences at the targeted AAVS1 locus (Figure 4B). This step in the procedure was also very efficient with Cre-mediated excision of these floxed cassettes occurring on average in >90% of integrated recombinant clones and was independent of the size of the DNA payload integrated (Figure 4C). Furthermore, as expected due to the use of different pairs of heterotypic lox sequences, long-range PCR across the integrated sequence indicated no genomic rearrangements had occurred (Figure 4D).

Overall when we combine serine recombinase-mediated integration of DNA vectors along with subsequent tyrosine recombinase-mediated excision of the supporting sequences we can generate targeted clonal lines within 6 weeks, irrespective of the size of the DNA integrated. Furthermore, due to the efficiency of the method of the invention when combined with the selection strategy, typically less than 10 single cell-derived clones require screening to identify multiple clonal hiPSCs that have the desired modification. This therefore also offers the possibility to integrate multiple different DNA payloads into the same genomic locus simultaneously and subsequently demultiplex these variants by single cell deposition.

4. Expediting the evaluation of optogenetic sensors and the generation of multi-parameter reporter hiPSCs

To demonstrate the utility and rapid adaptability of the method of the invention to generate a series of hiPSC lines carrying different combinations of transgenes in the same genetic locus, we introduced a set of genetic reporters to evaluate the complete excitation-contraction (EC) coupling cascade in hiPSC- CMs. These consisted of the genetically-encoded voltage indicator ASAP2f for assessing the cardiac action potential (Zhang et al. 2019), the genetically-encoded calcium indicator jRCaMP1b for measuring changes in cytosolic Ca?* levels (Dana et al. 2016), and a far-red fluorescent reporter fused to a plasma membrane-localisation signal (Lck-miRFP703) (Shcherbakova et al. 2016; Chertkova et al. 2020) to quantify contraction. To build the expression cassettes for each reporter, a modular cloning strategy (Weber et al. 2011; Martella et al. 2017) in which the basic components (e.g. promoter, localisation signal, coding sequence, terminator) could be correctly assembled in a one-step golden gate cloning reaction was employed. The resulting expression cassettes could then be assembled either individually (Figure 5A) or as a complex multi-unit circuit into a modified donor vector (Duportet et al. 2014) for subsequent integration into the AAVS1-Bxb1 acceptor hiPSC line. Initially each of these reporters were inserted individually (Figure 58).

Blasticidin selection followed by expression of Cre recombinase resulted in a set of targeted hiPSC lines, with PCR screening and Sanger sequencing confirming each of the reporters were targeted to the AAVS1 focus and that the “floxed” exogenous sequences were excised (Figure 5C). Additionally, fluorescence imaging confirmed that each of these hiPSC lines constitutively expressed the corresponding reporter {Figure 5D). All three fluorescent proteins were located in the expected cell compartment, with the ASAP2f and miRFP703 reporters localised to the cell membrane whereas expression of the jRCaMP1b reporter was found in the cytosol, due to the NES sequence. Similarly flow cytometry confirmed that >98% of the hiPSCs expressed the fluorescent reporter (Figure 5E).

The individual reporter lines were subsequently differentiated into hiPSC-CMs (Figure 5F) that displayed characteristic sarcomeric structures that were positive for a-actinin (Figure 5G}. The constitutive expression of the reporters was also evaluated in the hiPSC-CMs by microscopy and flow cytometry, with each of the reporters remaining localised to the expected subcellular regions and being expressed in >95% of the differentiated cells (Figure 5H & 51). Furthermore, each of these fluorescent sensors were evaluated for their ability to optically measure the action potential (AP), cytosolic Ca?’ levels and contraction of hiPSC-CMs (Figure 51). Indeed, hiPSC-CMs expressing ASAP2f showed the expected periodic changes in fluorescence intensity, with a reduction detected in the depolarization phase, followed by an increase during membrane repolarization and the diastolic resting phase. Likewise, hiPSC-CMs expressing jRCaMP1b displayed cyclic changes in fluorescence, with an increase in fluorescence intensity during the systolic rise in intracellular calcium levels, followed by a reduction in fluorescence during relaxation. Finally, hiPSC-CMs expressing miRFP703 showed modulating changes in fluorescence that allowed for quantification of contraction using the software, MUSCLEMOTION (Sala et al. 2018).

Based on this we then proceeded to build a donor vector comprising of all three reporters plus an additional copy of ASAP2f to increase its expression levels in the hiPSC-CMs (Figure 6A). Co-transfection of the donor and Bxb1 expression vectors followed by blasticidin selection resulted in a hiPSC population in which ~35% of the cells had integrated the 19.5 kb multi-reporter construct (Figure 68). Transfection with Cre recombinase and clonal isolation resulted in an hiPSC line (hereafter referred to as AAVS1- AJMA) that was correctly targeted and with no internal rearrangements of the transgenes (Figure 6C & 6D). Furthermore, the AAVSI-AIMA hiPSCs co-expressed all 3 reporters in >90% of the cells, with a stronger ASAP2f signal evident (Figure GE & 6F).

We subsequently differentiated the AAVS1-AJMA hiPSCs to cardiomyocytes using a small molecule differentiation protocol (Figure 5F). While all 3 reporters were still clearly expressed in the hiPSC-CMs and the proportion of cells expressing ASAP2f was unchanged, we did observe some silencing of jRCaMP1b and Ick-miRFP703 (Figure 6G & 6H). The addition of insulator sequences between each reporter could possibly reduce this effect. Despite this, it was still possible to obtain measurements for the EC coupling cascade using these sensors (Figure 61}.

Furthermore, the expected drug-induced changes in AP (E-4031-induced prolongation), intracellular Ca?* transients (verapamil-induced amplitude reduction}, and contraction (isoprenaline-induced duration shortening) were detected by these reporters (Figure 6J-1).

Together, these results demonstrate the ease with which the procedure enables different genetic sensors to be rapidly integrated into a specific genetic locus. Furthermore, by adapting the integration vector construct for modular cloning and developing a library of components for one-step construction of expression cassettes, this allowed for easy assembly of the different reporters into a 14 kb multi-unit construct comprising of all 3 sensors. We validated that the resulting AAVS1-AJMA hiPSCs could subsequently provide an optical readout of EC coupling processes in hiPSC-CMs and be used to assess drug-induced electrophysiological, calcium handling and mechanical changes. The stable integration of these reporters also reduces experimental variability and allows repeated measurements of the same hiPSC-CMs over multiple days to evaluate chronic pharmacological responses.

5. The simultaneous generation of a panel of isogenic hiPSCs harboring a range of KCNH2 variants Lastly, we investigated the possibility to generate rapidly and simultaneously a library of genetic variants in the gene, KCNH2, using the method of invention.

Heterozygous mutations in KCNH2, which encodes the hERG ion channel, can lead to either long QT syndrome type 2 (LQT2), short QT type 1, or Brugada syndrome (Chen et al. 2016). However, there are also rare variants identified within this gene that do not affect the functionality of the encoded ion channel {Ng et al. 2020). Because there appears to be no limitation on the size of the DNA sequence that can inserted using the method, we opted to replace the entire KCNH2 genomic locus (50 kb) on one allele with the Bxb1-LP (Figure 7A). This meant that not only could KCNH2 coding variants be introduced in heterozygosity in a nearly identical genomic context to that in individuals with the variant, but could also enable common single nucleotide polymorphisms (SNPs} identified within non-coding regions of the KCNH2 locus to be examined.

We confirmed that the resulting hiPSC line (KCNH2*4<) was correctly targeted, contained a single integration of the LP and was monoallelic for KCNH2 (Figure 7B & 7C). To confirm that we could efficiently re-insert KCNH2 back into its genomic locus, we first reintroduced the KCNH2 wild type sequence into the KCNH27A< hiPSC line. ~30% had undergone both the integration and excision steps, showing a similar efficiency to that observed for the AAVS1 locus and indicating the procedure was not locus dependent. ddPCR also confirmed that for one of the resulting hiPSC lines (KCNH274<") 2 copies of KCNH2 were now present while eGFP and bsdR were removed (Figure 7C). Based on this, we investigated whether the method could facilitate the multiplex insertion of individual missense KCNH2 variants into a pool of cells (Figure 7D). For this we focused on mutations identified in exon 7 of KCNH2 which encodes the pore region of hERG.

We selected 12 variants and using recombineering first separately introduced these mutations into a BAC vector containing the KCNH2 gene, and then subcloned the modified gene into the Bxb1-donor vector as the payload to insert.

The resulting vectors were pooled and transfected along with Bxb1 recombinase into <1x10° KCNH27/AC hiPSCs (e.g. one well of a 6-well plate). Modifications to the enrichment step improved the proportion of recombined hiPSCs to 66.5% (Figure 7E}, with all 12 variants detected within this population (Figure 8). Following Cre-mediated excision of the auxiliary sequences, 11 out of the 12 variants were recovered following screening of 208 colonies {Figure 8), with the entire procedure taking ~2 months.

We further characterized an hiPSC clone (KCNH27A<A%61T} heterozygous for the KCNH2 variant A561T (Figure 7F). hiPSC-CMs that carry this mutation exhibit a prolonged field potential duration (FPD) (Matsa et al. 2011; Brandäo et al. 2020), reflective of the electrophysiological characteristics of Long QT syndrome type 2. Likewise, the KCNH2*/A<A%IT hipsC-CMs had a pronounced FPD increase compared to the KCNH27A<Wt hipsC-CMs (Figure 7G), confirming that KCNH2 variant models generated using the method of invention also manifest the expected disease phenotype.

These results demonstrate how the gene editing procedure of the invention also can be used as a high- throughput method to multiplex and simultaneous insert potentially hundreds of different disease- linked variants into a control hiPSC line for downstream evaluation all on the same genetic background. Such panels could be used to test the efficacy of pharmacological compounds against individual mutations {e.g. personalized medicine). For example, nearly 500 mutations in KCNH2 have been associated with LQT2 with at least 170 of these predicted to cause trafficking defects (Anderson et al. 2014). Recently it has been proposed that lumacaftor, an FDA-approved drug to treat cystic fibrosis, could be re-purposed as a potential therapeutic for patients with LQT2 caused by trafficking defective variants (Schwartz et al. 2019). However in some cases the drug appears to cause an opposite effect (O'Hare et al. 2020), indicating that large scale efficacy studies of the drug using a large cohort of KCNH2 variant hiPSC-CMs will likely be required to advance this further in the clinic.

Another application that would benefit from this multiplex approach is in the classification of rare variants. Large scale genetic sequencing projects have revealed that rare variants are prevalent in the general population. The difficulty in distinguishing pathogenic variants from rare benign variants when performing genetic testing for inherited disorders has resulted in large proportions of patients having variants classified as being of “uncertain significance” and so not clinically actionable. Therefore, platforms that are rapid and do not require an individualized targeting strategy for each variant would be highly advantageous in a diagnostic setting. A previous study investigated whether DICE could be used to generate such a hiPSC panel of TNNT2 variants (Lv et al. 2018). While they were able to isolate heterozygous clones for 14 coding variants, their reported efficiency was ~5%, and they only obtained lines for 712% of the variants they tried to introduce. Additionally, the procedure was restricted to integrating a DNA payload of < 1 kb and so only a partial cDNA spanning TNNT2 exons 6 to 17 could be integrated, thereby potentially resulting in the loss of regulatory elements that control the expression of the variant. Although we have modified a different genetic locus, we report a much higher targeting efficiency (734%) and as well recover ~92% of the variants introduced. Furthermore, with the method of the invention, non-coding genomic regions are retained in the integration. This also permits SNPs identified in genome-wide association studies that might influence the disease phenotype to be modified and investigated.

SEQUENCES The following longer sequences are used in the examples, the full sequences of which are provided in the accompanying sequence listing which is incorporated by reference herein: >pENTR-eGFP-aitP{bxb}-*bsdR plasmid (SEQ ID NO 179) >pENTR-mCherry-attP{PhiC31}-*BleoR plasmid (SEQ ID NO 180)

> AAVS1-Bxb1-LP-TC plasmid (SEQ ID NO 181) > AAVS1-PhiC31-LP-TC plasmid (SEQ ID NO 182) > pBR_attB(bxb)_lox plasmid (SEQ ID NO 183) >pl5_attB(bxb) fox plasmid (SEQ ID NO 184)

> pBR_attB(bxb)_ccdB_lox plasmid (SEQ ID NO 185) >pBR_attB(C31)_FRT plasmid (SEQ ID NO 186) > Test Sequence of DNA payload (2 kb) (SEQ ID NO 187) > Test Sequence of DNA payload (5 kb} (SEQ ID NO 188) > Test Sequence of DNA payload (10 kb) (SEQ ID NO 189)

> Test Sequence of DNA payload (15 kb) (SEQ ID NO 190) > Sequence of Bxb1_attB fragment introduced into Bac RP11-10L20 by recombineering (SEQ ID NO 191) > pBR-attB_ASAP2f plasmid (SEQ ID NO 192) > pBR-attB_jRCaMP1b plasmid (SEQ ID NO 193) > pBR-attB_Lck-miRFP703 plasmid (SEQ ID NO 194)

> pBR-attB_AJMA_donor plasmid (SEQ ID NO 195) > KCNH2-Bxb1-LP-TC plasmid (SEQ ID NO 196) > p15-attB_KCNH2_wt_donor plasmid (SEQ ID NO 197) >pEfBOS_flpO_2A_crelRESPuro plasmid (SEQ ID NO 198) >Sequence of the genomic region surrounding the deletion of KCNH2 (SEQ ID NO 199)

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P318031NL_ST25{9969840} (1)

SEQUENCE LISTING <110> Academisch Ziekenhuis Leiden h.o.d.n. LUMC <120> GENOMIC INTEGRATION <130> P318031INL <160> 203 <170> PatentIn version 3.5 <21e> 1 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> first spacer <400> 1 catat 5 <2105 2 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> first spacer <400> 2 gcgc 4 <2105 3 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> BxB1 attP recombination site and the first spacer <400> 3 ggtttgtctg gtcaaccacc gcggtctcag tggtgtacgg tacaaaccca tat 53 <2105 4 <211> 54 <212> DNA <213> Artificial Sequence Pagina 1

P318031NL_ST25{9969840} (1) <220> <223> phage C31 attP recombination site and the first spacer <400> 4 gtagtgcccc aactggggta acctttgagt tctctcagtt gggggcgtag gcgc 54 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gRNA landing pad in intron 1 of PPP1R12C <400> 5 ggggccacta gggacaggat 20 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> start codon, second spacer and attB recombination site <400> 6 atggccggct tgtcgacgac ggcggtctcc gtcgtcagga tcatcc 46 <210> 7 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> start codon, second spacer and phage C31 attB recombination site <400> 7 atggcctcga agccgcggtg cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac 60 tccacctcac ccatc 75 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <220>

Pagina 2

P318031NL_ST25{9969840} (1) <223> start codon, second spacer, attL recombination scar, and first spacer <400> 8 atggccggct tgtcgacgac ggcggtctca gtggtgtacg gtacaaaccc atat 54 <210> 9 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> start codon, second spacer, attL recombination scar, and first spacer <400> 9 atggcctcga agccgcggtg cgggtgccag ggcgtgccct tgagttctct cagttggggs 60 cgtaggcgc 69 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pENTR HiFi rev <400> 10 gactggccgt cgttttac 18 <210> 11 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> lox251 pENTR HiFi Fwd <400> 11 ataacttcgt ataggagact ttatacgaag ttaatggatc ccctatagtg agtcg 55 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> Pagina 3

P318031NL_ST25{9969840} (1) <223> pENTR_loxP_HiFi_Fwd <400> 12 cgacgttgta aaacgacggc cagtcgctag cgagctcggc gcg 43 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PGK_GFP_HiFi_Rev <400> 13 gctcaccatg gtggcctgca ggtcgaaagg cccg 34 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> PGK_GFP_HiFi_Fwd <400> 14 tttcgacctg caggccacca tggtgagcaa gggcgag 37 <210> 15 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> bxb_GFP_HiFi_Rev <400> 15 gacaaaccac gtatatctag acatgataag atacattgat gagtttgg 48 <210> 16 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> SV40 bxb_HiFi_Fwd <400> 16 tcttatcatg tctagatata cgtggtttgt ctggtcaacc accgcggtct cagtggtgta 60 Pagina 4

P318031NL_ST25{9969840} (1) cggtacaaac ccatatgcca agcctttgtc tcaag 95 <210> 17 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> lox251 bpA HiFi Rev <400> 17 tgtaatacga ctcactatag gggatccatt aacttcgtat aaagtctcct atacgaagtt 60 atgcctcaga agccatagag 80 <210> 18 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> lox_eGFP attP(Bxb) lox251 Fwd <400> 18 ctccacccca cagtggggca agtcacgacg ttgtaaaacg acg 43 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> lox_eGFP attP(Bxb}) lox251 Rev <400> 19 accaatcctg tccctagtaa tgaccatgta atacgactca ctataggg 48 <210> 20 <211> 241 <212> DNA <213> Artificial Sequence <220> <223> bxb attB lox gBlock <400> 20 ccatttcagg tgtcgtgagg aattcgccac catggccggc ttgtcgacga cggcggtctc 60 Pagina 5

P318031NL_ST25{9969840} (1) cgtcgtcagg atcatccgga tccataactt cgtatagcat acattatacg aagttatcat 120 gatattcggc aagcaggcat cgactagtta attaagctag cgctgcaaga actcttcctc 180 acgataactt cgtataggag actttatacg aagttaagcg ctcactggcc gtcgttttac 240 a 241 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pENTR HiFi rev <400> 21 gactggccgt cgttttac 18 <210> 22 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> bleo_FRT-pENTR HiFi_ Fwd <400> 22 gaagttccta ttccgaagtt cctattcttc aaatagtata ggaacttcga acatccagct 60 gatatcccc 69 <210> 23 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> pENTR_FRT_HiFibuild_Fwd <400> 23 cgacgttgta aaacgacggc cagtcttaat taagaagttc ctattccgaa gttc 54 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence

Pagina 6

P318031NL_ST25{9969840} (1) <220> <223> PGK cherry HiFi Rev <400> 24 tcaccatggt ggcaattgtc gaaaggcccg gagat 35 <210> 25 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PGK cherry HiFi Fwd <400> 25 cgggcctttc gacaattgcc accatggtga gcaagggcga g 41 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> (C31 cherry HiFi Rev <400> 26 gttggggcac tacggatcca tagagcccac cgca 34 <210> 27 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> (C31 bleo HiFibuild Fwd <400> 27 tgggctctat ggatccgtag tgccccaact ggggtaacct ttgagttctc tcagttgggg 60 gcgtaggcgc gccaagttga ccagtgcc 88 <210> 28 <211> 105 <212> DNA <213> Artificial Sequence <220>

Pagina 7

P318031NL_ST25{9969840} (1) <223> pENTR_F3_HiFi_Rev <400> 28 cactataggg gatatcagct ggatgttcga agttcctata ctatttgaag aataggaact 60 tcggaatagg aacttcataa gatacattga tgagtttgga caaac 105 <210> 29 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> FRT_pENTR _mCherry attP(?C31) F3 Fwd <400> 29 ctccacccca cagtggggca agtcacgacg ttgtaaaacg acg 43 <210> 30 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> FRT_pENTR _mCherry attP(?C31) F3 Rev <400> 30 accaatcctg tccctagtaa tgaccatgta atacgactca ctataggg 48 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C31_ attB_Fwd <400> 31 actccacctc acccatcgga tc 22 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BamHI_puroRev153

Pagina 8

P318031NL_ST25{9969840} (1) <400> 32 ggatccgtga ggaagagttc ttgcagc 27 <210> 33 <211> 185 <212> DNA <213> Artificial Sequence <220> <223> C31 attB_F3 gBlock <400> 33 tgtaaaacga cggccagtga gcgctgaagt tcctatacta tttgaagaat aggaacttcg 60 gaataggaac ttcggatccg atgggtgagg tggagtacgc gCCCggggag cccaagggca 120 cgccctggca cccgcaccgc ggcttcgagg ccatggtggc gaattcctca cgacacctga 180 aatgg 185 <210> 34 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> FRT gBlock <400> 34 actccacctc acccatcgga tccgaagttc ctattccgaa gttcctattc tctagaaagt 60 ataggaactt catgatattc ggcaagcagg catcgactag ttaattaagc tagcgctgca 120 agaactcttc ctcacggatc c 141 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pENTR HiFi rev <400> 35 gactggccgt cgttttac 18 <210> 36 <211> 69

Pagina 9

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> bleo_FRT-pENTR HiFi_ Fwd <400> 36 gaagttccta ttccgaagtt cctattcttc aaatagtata ggaacttcga acatccagct 60 gatatcccc 69 <210> 37 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> p15-SV49 HiFi_Fwd <400> 37 atggttgctg actaattgag atgcagcgct agcggagtgt atactg 46 <210> 38 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> amp-lox251 HiFi Rev <400> 38 taatagcgaa gaggcccgca ccgatacgtc aggtggcact tttcg 45 <210> 39 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> NdeI-BpiI-EFla_Fwd <400> 39 catatgaaga caatgccatt ggctccggtg cccgtc 36 <210> 40 <211> 42 <212> DNA <213> Artificial Sequence

Pagina 10

P318031NL_ST25{9969840} (1) <220> <223> NdeI-BpiIl neo4 Rev <400> 40 catatgaaga caagctacga tgcctgcttg ccgaatatca tg 42 <210> 41 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> XhoI-Bpil-Esp3I lacZ Fwd <400> 41 ctcgagaaga caatagcaga gacgacaggt ttcccgactg gaaagc 46 <210> 42 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> XhoI-BpilI-Esp3I-lacZ Rev <400> 42 ctcgagaaga caactccaga gacggtgtcg gggctggctt aac 43 <210> 43 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> XhoI-Bpil puro153 Fwd <400> 43 ctcgagaaga caaggagctg caagaactct tcctcacg 38 <210> 44 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> XhoI-BpiI_M13 Rev

Pagina 11

P318031NL_ST25{9969840} (1) <400> 44 ctcgagaaga caatccctgt aaaacgacgg ccagt 35 <210> 45 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Hpal-Esp3I-BpiI-ccdB Fwd <400> 45 gttaacgtct cctagctgcc tagtcttcac acataaccag gaggtcag 48 <210> 46 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> HpaI-Esp3I-BpilI-ccdB Rev <400> 46 gttaacgtct cactcctccc atgtcttctg aagtcagccc catacgat 48 <210> 47 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> BAC EFla recom Fwd <400> 47 catccgatgc aagtgtgtcg ctgtcgacgg tgaccctata gtcgagggac cttaattccc 60 tccccagce 68 <210> 48 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> BAC SV40 recom Rev <400> 48 gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag gatccagaca 60 Pagina 12

P318031NL_ST25{9969840} (1) tgataagata cattg 75 <210> 49 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_2kb_recom-Fwd <400> 49 aagatgctga tgactatgaa taataaataa ttatcctgag gagaactcca gctgcaagaa 60 ctcttcctca cg 72 <210> 50 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_2kb_recom-Rev <400> 50 gtagcagctg caggacagtg gccatgtctg cactcagccg ggtctccagc cgatgcctgc 60 ttgccgaata tcatg 75 <210> 51 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_5kb recom-Rev <400> 51 aggtgcaggc agatgtcagc ctgcaggcac tcagggaagc ccttcagcac cgatgcctgc 60 ttgccgaata tcatg 75 <210> 52 <211> 75 <212> DNA <213> Artificial Sequence <220>

Pagina 13

P318031NL_ST25{9969840} (1) <223> KCNH2_10kb_recom-Rev <400> 52 ctgggccgca gagcccctgt cctgctcgcc ttcccggctg gggccgccat cgatgcctgc 60 ttgccgaata tcatg 75 <210> 53 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_15kb_recom-Rev <400> 53 catcctcgtt cttcacgggc accacatcca ccagacatag gaagcagctc ccgatgcctg 60 cttgccgaat atcatg 76 <210> 54 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_56kb recom Fwd <400> 54 ctagcacctg gaggtgtgtg aggggccagg atggactgcg gatagaccag tcgctgcaag 60 aactcttcct cacg 74 <210> 55 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_58kb recom Rev <400> 55 aagggggcag ggcctgtcag atggatccct gacaaccatc cgtctcaagt cgatgcctgc 60 ttgccgaata tcatg 75 <210> 56 <211> 98

Pagina 14

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> KCNH2_86b_M13 Fwd <400> 56 aggtgccctg tcttgggctc tgaaggccct acatacaaag tcatcctatc tgctgaggca 60 aagatataag gttacctcgt gtaaaacgac ggccagtc 98 <210> 57 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> KCNH2 80bp_lox251 Rev <400> 57 catttacatc tgggccacag tgtccctccg tctccctctg gccccggagc acatggccat 60 ctggttgtgc tgactgtgct gctatagggg atccattaac ttcg 104 <210> 58 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_ex7-amp_rec_Fwd <400> 58 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctagcgcttt 60 gtttattttt c 71 <210> 59 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> KCNH2 ex7-ccdB_rec_Rev <400> 59 tggcccggct agcagcctca gtttcctcca acttgggttc ctccaccgtg agccccatac 60 gatataagtt g 71 Pagina 15

P318031NL_ST25{9969840} (1) <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_ex7_Fwd <400> 60 cttgcccccc ttgccccatc 20 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_ex7_Rev <400> 61 tggcccggct agcagcctc 19 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (Bxbl) targeting - 5' junction forward primer <400> 62 ccggaactct gccctctaa 19 <210>5 63 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (Bxbl) targeting - 5' junction reverse primer <400> 63 ccatgatata gacgttgtgg ctgttg 26 <210> 64 <211> 23

Pagina 16

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (Bxbl) targeting - 3' junction forward primer <400> 64 cggcagttgg gattcgtgaa ttg 23 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (Bxbl) targeting - 3' junction reverse primer <400> 65 gtgagtttgc caagcagtca 20 <210> 66 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (C31) targeting - 5' junction forward primer <400> 66 ccggaactct gccctctaa 19 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (C31) targeting - 5' junction reverse primer <400> 67 ccgtcctcga agttcatcac 20 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220>

Pagina 17

P318031NL_ST25{9969840} (1) <223> AAVS1 acc (C31) targeting - 3' junction forward primer <400> 68 cccacaacga ggactacacc atc 23 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AAVS1 acc (C31) targeting - 3' junction reverse primer <400> 69 gtgagtttgc caagcagtca 20 <210> 70 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Bxbl Donor vector integration (attR) - AAVS1 forward primer <400> 70 caagatccgc cacaacatcg aggac 25 <21e> 71 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Bxbl Donor vector integration (attR) - AAVS1 reverse primer <400> 71 cgatgcctgc ttgccgaata tcatg 25 <2105 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Bxbl Donor vector integration (attL) - AAVS1 forward primer <400> 72 ttttggagta cgtcgtcttt agg 23 Pagina 18

P318031NL_ST25{9969840} (1) <2105 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Bxbl Donor vector integration (attL) - AAVS1 reverse primer <400> 73 ctggcaacta gaaggcacag tcg 23 <210> 74 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Bxbl Donor vector integration (attP) - AAVS1 forward primer <400> 74 caagatccgc cacaacatcg aggac 25 <21e> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Bxbl Donor vector integration (attP) - AAVS1 reverse primer <400> 75 ctggcaacta gaaggcacag tcg 23 <210> 76 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> (C31 Donor vector integration (attR) - AAVS1 forward primer <400> 76 catggtgagc aagggcgagg ag 22 <21e> 77 <211> 22

Pagina 19

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> (C31 Donor vector integration (attR) - AAVS1 reverse primer <400> 77 agtcacgacg ttgtaaaacg ac 22 <21e> 78 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> (C31 Donor vector integration (attL) - AAVS1 forward primer <400> 78 ttttggagta cgtcgtcttt agg 23 <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> (C31 Donor vector integration (attL) - AAVS1 reverse primer <400> 79 gtgaaatttg tgatgctatt gc 22 <210> 80 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> (C31 Donor vector integration (attP) - AAVS1 forward primer <400> 80 catggtgagc aagggcgagg ag 22 <21e> 81 <211> 20 <212> DNA <213> Artificial Sequence <220>

Pagina 20

P318031NL_ST25{9969840} (1) <223> (C31 Donor vector integration (attP) - AAVS1 reverse primer <400> 81 cggaccacac cggcgaagtc 20 <210> 82 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BAC vector integration (attR) - AAVS1 forward primer <400> 82 gtgctgctgc ccgacaacca ctac 24 <210> 83 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BAC vector integration (attR) - AAVS1 reverse primer <400> 83 gaacctgcgt gcaatccatc tt 22 <210> 84 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BAC vector integration (attL) - AAVS1 forward primer <400> 84 ttttggagta cgtcgtcttt agg 23 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAC vector integration (attL) - AAVS1 reverse primer <400> 85 gtgagtttgc caagcagtca 20 Pagina 21

P318031NL_ST25{9969840} (1) <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - 2-15 kb payload forward primer <400> 86 ctcggcgcgc cataacttcg 20 <21e> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - 2-15 kb payload reverse primer <400> 87 gtgagtttgc caagcagtca 20 <210> 88 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1oxP/FRT cassette excision - AAVS1 forward primer <400> 88 ccggaactct gccctctaa 19 <210> 89 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 1oxP/FRT cassette excision - AAVS1 reverse primer <400> 89 cgatgcctgc ttgccgaata tcatg 25 <210> 90 <211> 22

Pagina 22

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> lox251/F3 cassette excision - AAVS1 forward primer <400> 90 gctgcaagaa ctcttcctca cg 22 <21e> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lox251/F3 cassette excision - AAVS1 reverse primer <400> 91 gtgagtttgc caagcagtca 20 <210>5 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - ASAP2f forward primer <400> 92 ttcgggtcac ctctcactcc 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - ASAP2f reverse primer <400> 93 ctcacagtcg tctccatggt 20 <210> 94 <211> 23 <212> DNA <213> Artificial Sequence <220>

Pagina 23

P318031NL_ST25{9969840} (1) <223> Optogenetic lox251 cassette excision - ASAP2f forward primer <400> 94 caacattctg gggcataaac tgg 23 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic lox251 cassette excision - ASAP2f reverse primer <400> 95 ggctccatcg taagcaaacc 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - jRCaMPlb forward primer <400> 96 ttcgggtcac ctctcactcc 20 <21e> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - jRCaMPlb reverse primer <400> 97 ttacgtcgcg atgagtcgac 20 <210> 98 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic lox251 cassette excision - jRCaMPlb forward primer <400> 98 ggtcaggtaa actacgaaga g 21 Pagina 24

P318031NL_ST25{9969840} (1) <210> 99 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic lox251 cassette excision - jRCaMPlb reverse primer <400> 99 ggctccatcg taagcaaacc 20 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - miRFP703 forward primer <400> 100 ttcgggtcac ctctcactcc 20 <21e> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - miRFP703 reverse primer <400> 101 ttcgcaattc gaatgagagg c 21 <21e> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic lox251 cassette excision - miRFP7@3 forward primer <400> 102 gcttcatccg tttcgagctg 20 <21e> 103 <211> 20

Pagina 25

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> Optogenetic lox251 cassette excision - miRFP783 reverse primer <400> 103 ggctccatcg taagcaaacc 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - AJMA forward primer <400> 104 ttegggtcac ctctcactcc 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic loxP cassette excision - AJMA reverse primer <400> 105 ctcacagtcg tctccatggt 20 <210> 106 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Optogenetic lox251 cassette excision - AJMA forward primer <400> 106 caacattctg gggcataaac tgg 23 <21e> 107 <211> 20 <212> DNA <213> Artificial Sequence <220>

Pagina 26

P318031NL_ST25{9969840} (1) <223> Optogenetic lox251 cassette excision - AJMA reverse primer <400> 107 ggctccatcg taagcaaacc 20 <21e> 108 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - ASAP2f-jRCaMP1b forward primer <400> 108 caacattctg gggcataaac tgg 23 <21e> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - ASAP2f-jRCaMPlb reverse primer <400> 109 ttacgtcgcg atgagtcgac 20 <21e> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - jRCaMPlb-miRFP763 forward primer <400> 110 ggtcaggtaa actacgaaga g 21 <21e> 111 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - jRCaMPlb-miRFP7983 reverse primer <400> 111 ttcgcaattc gaatgagagg c 21 Pagina 27

P318031NL_ST25{9969840} (1) <21e> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - miRFP703-ASAP2f forward primer <400> 112 gcttcatccg tttcgagctg 20 <21e> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Integration integrity - miRFP703-ASAP2f reverse primer <400> 113 ctcacagtcg tctccatggt 20 <21e> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KCNH2+/Acc targeting - 5' junction forward primer <400> 114 acccggtcct tggtgttttc 20 <210> 115 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> KCNH2+/Acc targeting - 5' junction reverse primer <400> 115 ccatgatata gacgttgtgg ctgttg 26 <21e> 116 <211> 23

Pagina 28

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> KCNH2+/Acc targeting - 3' junction forward primer <400> 116 cggcagttgg gattcgtgaa ttg 23 <21e> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KCNH2+/Acc targeting - 3' junction reverse primer <400> 117 atttccaatc ctcaggggcg 20 <21e> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Donor vector integration (attR) - KCNH2 forward primer <400> 118 acccggtcct tggtgttttc 20 <21e> 119 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Donor vector integration (attR) - KCNH2 reverse primer <400> 119 cgatgcctgc ttgccgaata tcatg 25 <21e> 120 <211> 22 <212> DNA <213> Artificial Sequence <220>

Pagina 29

P318031NL_ST25{9969840} (1) <223> Donor vector integration (attL) - KCNH2 forward primer <400> 120 gctgcaagaa ctcttcctca cg 22 <21e> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Donor vector integration (attL) - KCNH2 reverse primer <400> 121 atttccaatc ctcaggggcg 20 <21e> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> loxP cassette excision - KCNH2 forward primer <400> 122 acccggtcct tggtgttttc 20 <21e> 123 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> loxP cassette excision - KCNH2 reverse primer <400> 123 cgatgcctgc ttgccgaata tcatg 25 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> lox251 cassette excision - KCNH2 forward primer <400> 124 gctgcaagaa ctcttcctca cg 22 Pagina 30

P318031NL_ST25{9969840} (1) <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lox251 cassette excision - KCNH2 reverse primer <400> 125 atttccaatc ctcaggggcg 20 <21e> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Screen KCNH2 exon 7 variants forward primer <400> 126 caaggaggca ggtggtgtag 20 <21e> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Screen KCNH2 exon 7 variants reverse primer <400> 127 cctccaactt gggttcctcc 20 <21e> 128 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> eGFP forward primer <400> 128 gccgacaagc agaagaacg 19 <21e> 129 <211> 20

Pagina 31

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> eGFP reverse primer <400> 129 gggtgttctg ctggtagtgg 20 <21e> 130 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> eGFP probe <400> 130 agatccgcca caacatcgag g 21 <21e> 131 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BsdR forward primer <400> 131 tggcaacctg acttgtatcg 20 <21e> 132 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BsdR reverse primer <400> 132 gtccatcact gtccttcact atc 23 <21e> 133 <211> 24 <212> DNA <213> Artificial Sequence <220>

Pagina 32

P318031NL_ST25{9969840} (1) <223> BsdR Probe <400> 133 cgacaggtgc ttctcgatct gcat 24 <210> 134 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mCherry forward primer <400> 134 gtgagcaagg gcgaggag 18 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mCherry reverse primer <400> 135 ctcgatctcg aactcgtggc 20 <21e> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mCherry Probe <400> 136 tcaaggtgca catggagggc 20 <21e> 137 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> BleoR forward primer <400> 137 agttgaccag tgccgttcc 19 Pagina 33

P318031NL_ST25{9969840} (1) <21e> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BleoR reverse primer <400> 138 cgaagtcgtc ctccacgaag 20 <21e> 139 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BleoR Probe <400> 139 agccggtcgg tccagaac 18 <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Bxbl-attP forward primer <400> 140 gcattctagt tgtggtttgt cc 22 <21e> 141 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Bxbl-attP reverse primer <400> 141 atgagggtgg attcttcttg ag 22 <21e> 142 <211> 21

Pagina 34

P318031NL_ST25{9969840} (1) <212> DNA <213> Artificial Sequence <220> <223> Bxbl-attP Probe <400> 142 cgtggtttgt ctggtcaacc a 21 <21e> 143 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Bxbl-attR forward primer <400> 143 gcattctagt tgtggtttgt cc 22 <210> 144 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Bxbl-attR reverse primer <400> 144 cttaattaac tagtcgatgc ctgc 24 <210> 145 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Bxbl-attR Probe <400> 145 tctccgtcgt caggatcatc c 21 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220>

Pagina 35

P318031NL_ST25{9969840} (1) <223> PhiC31-attP forward primer <400> 146 gaggattggg aagacaatag c 21 <21e> 147 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PhiC31-attP reverse primer <400> 147 aagtcgtcct ccacgaag 18 <21e> 148 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PhiC31-attP Probe <400> 148 catgctgggg atgcggtg 18 <210> 149 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PhiC31-attR forward primer <400> 149 gaggattggg aagacaatag c 21 <21e> 150 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PhiC31-attR reverse primer <400> 150 cctgcttgcc gaatatcatg 20 Pagina 36

P318031NL_ST25{9969840} (1) <21e> 151 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PhiC31-attR Probe <400> 151 atccgatggg tgaggtggag 20 <21e> 152 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> KCNH2 forward primer <400> 152 cttgccccat caacgg 16 <21e> 153 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KCNH2 reverse primer <400> 153 gcacactcac agccaatg 18 <210> 154 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_A561T Probe <220> <221> misc feature <222> (8)..(9) <223> locked nucleotide

Pagina 37

P318031NL_ST25{9969840} (1) <220> <221> misc feature <222> (11)..(11) <223> locked nucleotide <400> 154 cgctcatcac gcact 15 <210> 155 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_A558P Probe <220> <221> misc feature <222> (6)..(7) <223> locked nucleotide <400> 155 accttcccgc tca 13 <210> 156 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_A614V Probe <220> <221> misc feature <222> (6)..(8) <223> locked nucleotide <400> 156 tgacggtgct ctac 14 <21e> 157 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_H578P Probe

Pagina 38

P318031NL_ST25{9969840} (1) <220> <221> misc feature <222> (8)..(8) <223> locked nucleotide <220> <221> misc feature <222> (10)..(10) <223> locked nucleotide <220> <221> misc feature <222> (12)..(12) <223> locked nucleotide <400> 157 agcagccacc cat 13 <21e> 158 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_G584S Probe <220> <221> misc feature <222> (7)..(7) <223> locked nucleotide <220> <221> misc feature <222> (9)..(11) <223> locked nucleotide <400> 158 tcacgcatca gct 13 <210> 159 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_N588D Probe

Pagina 39

P318031NL_ST25{9969840} (1) <220> <221> misc feature <222> (10)..(11) <223> locked nucleotides <400> 159 tggctgcacg acct 14 <210> 160 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_N588K Probe <220> <221> misc feature <222> (5)..(5) <223> locked nucleotide <220> <221> misc feature <222> (8)..(10) <223> locked nucleotide <400> 160 ctgcacaagc tgg 13 <21e> 161 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_T618I Probe <220> <221> misc feature <222> (4)..(5) <223> locked nucleotide <220> <221> misc feature <222> (7)..(8) <223> locked nucleotide

Pagina 40

P318031NL_ST25{9969840} (1) <220> <221> misc feature <222> (11)..(11) <223> locked nucleotide <400> 161 acttcatctt cagc 14 <21e> 162 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_T618S Probe <220> <221> misc feature <222> (5)..(7) <223> locked nucleotide <220> <221> misc feature <222> (10)..(11) <223> locked nucleotide <400> 162 cttcagcttc agc 13 <21e> 163 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_F627L Probe <220> <221> misc feature <222> (3)..(4) <223> locked nucleotide <220> <221> misc feature <222> (6)..(6) <223> locked nucleotide <220>

Pagina 41

P318031NL_ST25{9969840} (1) <221> misc feature <222> (8)..(8) <223> locked nucleotide <400> 163 cttgggcaac gtc 13 <210> 164 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_G628S Probe <220> <221> misc feature <222> (4)..(6) <223> locked nucleotide <220> <221> misc feature <222> (9)..(9) <223> locked nucleotide <220> <221> misc feature <222> (12)..(12) <223> locked nucleotide <400> 164 ctttagcaac gtct 14 <210> 165 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> KCNH2_N633S Probe <220> <221> misc feature <222> (5)..(7) <223> locked nucleotide <220> <221> misc feature

Pagina 42

P318031NL_ST25{9969840} (1) <222> (12)..(12) <223> locked nucleotide <400> 165 tcccagcacc aac 13 <210> 166 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cas9-KCNH2 gRNA RNP complex (gRNA) <400> 166 ctggttgtgc tgactgtgct 20 <210> 167 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs121912516 gBlock <400> 167 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac cttcccgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 168 <211> 523 <212> DNA <213> Artificial Sequence

Pagina 43

P318031NL_ST25{9969840} (1) <220> <223> rs199472921 gBlock <400> 168 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcacgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 169 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs794728376 gBlock <400> 169 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacccatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 170

Pagina 44

P318031NL_ST25{9969840} (1) <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199473428 gBlock <400> 170 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcagctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 171 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199473431 gBlock <400> 171 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacgacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480

Pagina 45

P318031NL_ST25{9969840} (1) gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 172 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs194894021 gBlock <400> 172 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaagctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 173 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199472944 gBlock <400> 173 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggtgctcta cttcaccttc agcagcctca 360

Pagina 46

P318031NL_ST25{9969840} (1) ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 174 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199472947 gBlock <400> 174 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcatcttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 175 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199472947 gBlock <400> 175 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240

Pagina 47

P318031NL_ST25{9969840} (1) tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcagcttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 176 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199473039 gBlock <400> 176 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttgggcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 177 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs1219125987 gBlock <400> 177 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120

Pagina 48

P318031NL_ST25{9969840} (1) cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg ctttagcaac gtctctccca acaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 178 <211> 523 <212> DNA <213> Artificial Sequence <220> <223> rs199472961 gBlock <400> 178 cttgcccccc ttgccccatc aacggaatgt gccccttccc tgtcccccag ctgatcgggc 60 tgctgaagac tgcgcggctg ctgcggctgg tgcgcgtggc gcggaagctg gatcgctact 120 cagagtacgg cgcggccgtg ctgttcttgc tcatgtgcac ctttgcgctc atcgcgcact 180 ggctagcctg catctggtac gccatcggca acatggagca gccacacatg gactcacgca 240 tcggctggct gcacaacctg ggcgaccaga taggcaaacc ctacaacagc agcggcctgg 300 gcggcccctc catcaaggac aagtatgtga cggcgctcta cttcaccttc agcagcctca 360 ccagtgtggg cttcggcaac gtctctccca gcaccaactc agagaagatc ttctccatct 420 gcgtcatgct cattggctgt gagtgtgccc aggggeggge gECggggaga gcccacggtg 480 gaggaaccca agttggagga aactgaggct gctagccggg cca 523 <210> 179 <211> 4607 <212> DNA <213> Artificial Sequence <220> <223> pENTR-eGFP-attP(bxb)-*bsdR plasmid <400> 179

Pagina 49

P318031NL_ST25{9969840} (1) ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540 aacgacggcc agtcgctagc gagctcggcg cgccataact tcgtatagca tacattatac 600 gaagttagaa ttcgccttct gcaggagcgt acagaaccca gggccctggc acccgtgcta 660 ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct 720 gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag 780 gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttccactc ctcccctagt 840 caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc 900 acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct 960 ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag 1020 gggtgggtcc SggggCgggc tcaggggegg getcagggge BSEECggECg cccgaaggtc 1080 ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctce 1140 ttectcatct ccgggccttt cgacctgcag gccaccatgg tgagcaaggg cgaggagctg 1200 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1260 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1320 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 1380 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 14409 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 1500 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 1560 Pagina 50

P318031NL_ST25{9969840} (1) atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 1620 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 1680 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 1740 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 1800 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 1860 gggatcactc tcggcatgga cgagctgtac aagtaactgc agcgcgggga tctcatgctg 1920 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 1980 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 2040 aaactcatca atgtatctta tcatgtctag atatacgtgg tttgtctggt caaccaccgc 2100 ggtctcagtg gtgtacggta caaacccata tgccaagcct ttgtctcaag aagaatccac 2160 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt 2220 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt 2280 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg 2340 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg 2400 acggtgtcga caggtgcttc tcgatctgca tcctgggatc aaagcgatag tgaaggacag 2460 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga 2520 gggctaaatt accctgttat ccctaccteg actgtgcctt ctacagcctc gactgtgcct 2580 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 2640 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 2700 tgtcattcta ttctgggggg tggggtgggg Caggacagca agggggagga ttgggaagac 2760 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcata acttcgtata 2820 ggagacttta tacgaagtta atggatcccc tatagtgagt cgtattacat ggtcatagct 2880 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa 2940 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt 3000 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat 3060 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc 3120 Pagina 51

P318031NL_ST25{9969840} (1) ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 3180 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 3240 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 3300 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 3360 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 3420 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 3480 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 3540 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 3600 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 3660 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 3720 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 3780 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg 3840 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg 3900 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 3960 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4020 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4080 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4140 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4200 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4260 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4320 accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga 4380 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4440 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4500 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4560 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt 4607 <210> 180 Pagina 52

P318031NL_ST25{9969840} (1) <211> 4615 <212> DNA <213> Artificial Sequence <220> <223> pENTR-mCherry-attP(PhiC31)-*BleoR plasmid <400> 180 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540 aacgacggcc agtcttaatt aagaagttcc tattccgaag ttcctattct ctagaaagta 600 taggaacttc agcttgatat cgaattaatt ctaccgggta ggggaggcgc ttttcccaag 660 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct 720 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg 780 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct 840 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga 900 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg 960 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggeg gECLCaggEEg 1020 cgggctcagg BECggEECgEg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct 1080 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacaat 1140 tgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg 1200 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggs 1260 Cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc 1320

Pagina 53

P318031NL_ST25{9969840} (1) cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt 1380 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg 14409 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct 1500 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg 1560 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga 1620 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta 1680 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta 1740 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca 1800 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaat 1860 cgcgcgcgac tcgagccctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 1920 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 1980 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg LggggtggEgeg 2040 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 2100 tctatggatc cgtagtgccc caactggggt aacctttgag ttctctcagt tgggggcgta 2160 ggcgcgccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gCCggagcgg 2220 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg 2280 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg 2340 acaacaccct ggcctgggtg tgggtgegeg gcctggacga gctgtacgcc gagtggtcgg 2400 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc 2460 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg 2520 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa 2580 gettgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 2640 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 2700 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 2760 gtttgtccaa actcatcaat gtatcttatg aagttcctat tccgaagttc ctattcttca 2820 aatagtatag gaacttcgaa catccagctg atatccccta tagtgagtcg tattacatgg 2880 Pagina 54

P318031NL_ST25{9969840} (1) tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac 2940 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa 3000 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg 3060 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa 3120 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta 3180 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc 3240 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg 3300 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata 3360 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc 3420 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt 3480 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 3540 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct 3600 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag 3660 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 3720 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata 3780 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt 3840 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc 3900 ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 3960 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4020 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4080 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 4140 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4200 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4260 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4320 cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg 4380 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4440 Pagina 55

P318031NL_ST25{9969840} (1) gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 4500 gacttgagcg tcgatttttg tgatgctcgt Caggggggcg gagcctatgg aaaaacgcca 4560 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtt 4615 <210> 181 <211> 7955 <212> DNA <213> Artificial Sequence <220> <223> AAVS1-Bxb1-LP-TC plasmid <400> 181 ctagtcctgc aggtttaaac gaattcgccc tttgctttct ctgaccagca ttctctcccc 60 tgggcctgtg ccgctttctg tctgcagctt gtggcctggg tcacctctac ggctggccca 120 gatccttccc tgccgcctcc ttcaggttcc gtcttcctecc actcccetett ccccttgcte 180 tctgctgtgt tgctgcccaa ggatgctctt tccggagcac ttccttctcg gcgctgcacc 240 acgtgatgtc ctctgagcgg atcctccccg tgtctgggtc ctctccgggc atctctcctc 300 cctcacccaa ccccatgccg tcttcactcg ctgggttccc ttttccttct ccttctgggg 360 cctgtgccat ctctcgtttc ttaggatggc cttctccgac ggatgtctcc cttgcgtccc 420 gcctcccctt cttgtaggcc tgcatcatca ccgtttttct ggacaacccc aaagtacccc 480 gtctccctgg ctttagccac ctctccatcc tcttgctttec tttgcctgga caccccgttc 540 tcctgtggat tcgggtcacc tctcactcct ttcatttggg cagctcccct acccccctta 600 cctctctagt ctgtgctagc tcttccagcc ccctgtcatg gcatcttcca ggggtccgag 660 agctcagcta gtcttcttcc tccaacccgg gcccctatgt ccacttcagg acagcatgtt 720 tgctgcctcc agggatcctg tgtccccgag ctgggaccac cttatattcc cagggccggt 780 taatgtggct ctggttctgg gtacttttat ctgtcccctc caccccacag tggggcaagt 840 cacgacgttg taaaacgacg gccagtcgct agcgagctcg gcgcgccata acttcgtata 900 gcatacatta tacgaagtta gaattcgcct tctgcaggag cgtacagaac ccagggccct 960 ggcacccgtg ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt 1020 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca 1080

Pagina 56

P318031NL_ST25{9969840} (1) catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcca 1140 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac 1200 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga 1260 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc 1320 agaggctggg aaggggtggg tccgggggeg ggctcagggg Cgggctcagg gECgSEESCES 1380 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg 14409 cgctgttctc ctcttcctca tctccgggcc tttcgacctg caggccacca tggtgagcaa 1500 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 1560 cggccacaag ttcagcgtgt ccggecgaggg cgagggcgat gccacctacg gcaagctgac 1620 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 1680 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 1740 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 1800 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 1860 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 1920 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 1980 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 2040 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 2100 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 2160 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaac LgCagcgcgg 2220 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta 2280 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 2340 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tagatatacg tggtttgtct 2400 ggtcaaccac cgcggtctca gtggtgtacg gtacaaaccc atatgccaag cctttgtctc 2460 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg 2520 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca 2580 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg 2640 Pagina 57

P318031NL_ST25{9969840} (1) ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct 2700 tgagcccctg cggacggtgt cgacaggtgc ttctcgatct gcatcctggg atcaaagcga 2760 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg 2820 gttatgtgtg ggagggctaa attaccctgt tatccctacc tcgactgtgc cttctacagc 2880 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcectt 2940 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3000 ttgtctgagt aggtgtcatt ctattctggg gggtggggLg gggcaggaca gcaaggggga 3060 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3120 ataacttcgt ataggagact ttatacgaag ttaatggatc ccctatagtg agtcgtatta 3180 catggtcatt actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct 3240 tcctagtctc ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg 3300 tgacacaccc ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt 3360 acgatggagc cagagaggat cctgggaggg agagcttgge agggggtggg agggaagsss 3420 gggatgcgtg acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg 3480 agctgctctg acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac 3540 acttcccaag aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc 3600 taactttggc tcttcacctt tctagtcccc aatttatatt gttcctcegt gcgtcagttt 3660 tacctgtgag ataaggccag tagccagccc cgtcctggca gggctgtggt gaggagggEgEg 3720 gtgtccgtgt ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt 3780 ggccgcctct actccctttc tetttctcca tccttetttec cttaaagagt ccccagtgct 3840 atctgggaca tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg 3900 cttctgggtt tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt 3960 cctcgtccac catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct 4020 ctgctctaag ggcgaattcg cggccgctaa attcaattcg ccctatagtg agtcgtatta 4080 caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 4140 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 4200 Pagina 58

P318031NL_ST25{9969840} (1) cgatcgccct tcccaacagt tgcgcagcct atacgtacgg cagtttaagg tttacaccta 4260 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc 4320 geggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg 4380 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat 4440 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa 4500 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gcatgagatt 4560 atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg 4620 ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa 4680 gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg 4740 gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg 4800 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc 4860 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg 4920 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 4980 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 5040 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct 5100 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 5160 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 5220 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 5280 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 5340 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 5400 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatgcatg 5460 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 5520 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 5580 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 5640 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg 5700 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 5760 Pagina 59

P318031NL_ST25{9969840} (1) tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 5820 cattcaaata tgtatccgct catgagatta tcaaaaagga tcttcaccta gatcctttta 5880 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 5940 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6000 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6060 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6120 cagccagecg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6180 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6240 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6300 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6360 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6420 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6480 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6540 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6600 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6660 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6720 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6780 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 6840 tattgtctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 6900 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 6960 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 7020 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 7080 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 7140 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 7200 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 7260 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 7320 Pagina 60

P318031NL_ST25{9969840} (1) gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 7380 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 7440 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc agggggEgcgg 7500 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 7560 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 7620 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 7680 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7740 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7800 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7860 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 7920 tacgccaagc tcagaattaa ccctcactaa aggga 7955 <210> 182 <211> 7963 <212> DNA <213> Artificial Sequence <220> <223> AAVS1-PhiC31-LP-TC plasmid <400> 182 ctagtcctgc aggtttaaac gaattcgccc tttgctttct ctgaccagca ttctctcccc 60 tgggcctgtg ccgctttctg tctgcagctt gtggcctggg tcacctctac ggctggccca 120 gatccttccc tgccgcctcc ttcaggttcc gtcttcctecc actcccetett ccccttgcte 180 tctgctgtgt tgctgcccaa ggatgctctt tccggagcac ttccttctcg gcgctgcacc 240 acgtgatgtc ctctgagcgg atcctccccg tgtctgggtc ctctccgggc atctctcctc 300 cctcacccaa ccccatgccg tcttcactcg ctgggttccc ttttccttct ccttctgggg 360 cctgtgccat ctctcgtttc ttaggatggc cttctccgac ggatgtctcc cttgcgtccc 420 gcctcccctt cttgtaggcc tgcatcatca ccgtttttct ggacaacccc aaagtacccc 480 gtctccctgg ctttagccac ctctccatcc tcttgctttec tttgcctgga caccccgttc 540 tcctgtggat tcgggtcacc tctcactcct ttcatttggg cagctcccct acccccctta 600

Pagina 61

P318031NL_ST25{9969840} (1) cctctctagt ctgtgctagc tcttccagcc ccctgtcatg gcatcttcca ggggtccgag 660 agctcagcta gtcttcttcc tccaacccgg gcccctatgt ccacttcagg acagcatgtt 720 tgctgcctcc agggatcctg tgtccccgag ctgggaccac cttatattcc cagggccggt 780 taatgtggct ctggttctgg gtacttttat ctgtcccctc caccccacag tggggcaagt 840 cacgacgttg taaaacgacg gccagtctta attaagaagt tcctattccg aagttcctat 900 tctctagaaa gtataggaac ttcagcttga tatcgaatta attctaccgg gtaggggagg 960 cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct 1020 acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc 1080 cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc 1140 ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc 1200 tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc 1260 aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt gggLCCggEEg 1320 gcgggctcag gggcgggctc aggggCgggg cgggcgcccg aaggtcctcc ggaggcccgg 1380 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg 14409 gcctttcgac aattgccacc atggtgagca agggcgagga ggataacatg gccatcatca 1500 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga 1560 tCgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga 1620 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct 1680 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg 1740 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc 1800 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact 1860 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc 1920 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg 1980 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc 2040 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca 2100 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggegge atggacgagc 2160 Pagina 62

P318031NL_ST25{9969840} (1) tgtacaagta aatcgcgcgc gactcgagcc ctcgactgtg ccttctagtt gccagccatc 2220 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 2280 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 2340 gggtgggegte gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 2400 ggatgcggtg ggctctatgg atccgtagtg ccccaactgg ggtaaccttt gagttctctc 2460 agttgggggc gtaggcgcgc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac 2520 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag 2580 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac 2640 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac 2700 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc 2760 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 2820 gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc 2880 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 2940 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 3000 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 3060 cattctagtt gtggtttgtc caaactcatc aatgtatctt atgaagttcc tattccgaag 3120 ttcctattct tcaaatagta taggaacttc gaacatccag ctgatatccc ctatagtgag 3180 tcgtattaca tggtcattac tagggacagg attggtgaca gaaaagcccc atccttaggc 3240 ctcctccttec ctagtctcct gatattgggt ctaaccccca cctcctgtta ggcagattcc 3300 ttatctggtg acacaccccc atttcctgga gccatctctc tccttgccag aacctctaag 3360 gtttgcttac gatggagcca gagaggatcc tgggagggag agcttggcag ggggtgggag 3420 ggaagggggg gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct accctctccc 3480 agaacctgag ctgctctgac gcggctgtct ggtgcgtttc actgatcctg gtgctgcagc 3540 ttccttacac ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa taagttggtc 3600 ctgagttcta actttggctc ttcacctttc tagtccccaa tttatattgt tcctccgtgc 3660 gtcagtttta cctgtgagat aaggccagta gccagccccg tcctggcagg gctgtggtga 3720 Pagina 63

P318031NL_ST25{9969840} (1) geaggggggt gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct aggtgttcac 3780 caggtcgtgg ccgcctctac tccctttctc tttctccatc cttctttecct taaagagtcc 3840 ccagtgctat ctgggacata ttcctccgcc cagagcaggg tcccgcttcc ctaaggccct 3900 gctctgggct tctgggtttg agtccttggc aagcccagga gaggcgctca ggcttccctg 3960 tcccccttcc tegtccacca tctcatgccc ctggctctcc tgccccttcc ctacaggggt 4020 tcctggctct gctctaaggg cgaattcgcg gccgctaaat tcaattcgcc ctatagtgag 4080 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 4140 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 4200 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt 4260 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt 4320 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa 4380 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc 4440 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac 4500 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc 4560 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag 4620 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 4680 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 4740 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 4800 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 4860 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4920 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 4980 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 5040 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta 5100 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 5160 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 5220 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 5280 Pagina 64

P318031NL_ST25{9969840} (1) ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 5340 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 5400 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc 5460 catgcatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 5520 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 5580 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 5640 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat 5700 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 5760 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 5820 tctaaataca ttcaaatatg tatccgctca tgagattatc aaaaaggatc ttcacctaga 5880 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 5940 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 6000 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 6060 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 6120 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 6180 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 6240 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 6300 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 6360 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 6420 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 6480 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 6540 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 6600 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 6660 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 6720 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 6780 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 6840 Pagina 65

P318031NL_ST25{9969840} (1) atcagggtta ttgtctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 6900 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 6960 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 7020 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 7080 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 7140 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 7200 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 7260 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 7320 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 7380 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 7440 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 7500 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 7560 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 7620 ttaccgcctt tgagtgagct gataccgctc gCCgCagccg aacgaccgag cgcagcgagt 7680 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 7740 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 7800 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 7860 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 7920 accatgatta cgccaagctc agaattaacc ctcactaaag gga 7963 <210> 183 <211> 4813 <212> DNA <213> Artificial Sequence <220> <223> pBR_attB(bxb) lox plasmid <400> 183 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120

Pagina 66

P318031NL_ST25{9969840} (1) tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta 240 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 300 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 360 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 420 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc 480 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct 540 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 600 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggegeg 660 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 720 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 780 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc 840 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc 900 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg 960 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc 1020 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg 1080 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta 1140 ttteggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg 1200 CgaggCgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc 1260 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gCggcaaggc 1320 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag 1380 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa 14409 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag Laccgggcgc 1500 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggges 1560 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag 1620 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca 1680 Pagina 67

P318031NL_ST25{9969840} (1) ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa 1740 ttegccacca tggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccggatc 1800 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg 1860 actagttaat taagctagcg ctgcaagaac tcttcctcac gataacttcg tataggagac 1920 tttatacgaa gttaagcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 1980 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 2040 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 2100 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag 2160 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 2220 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttegettt cttcccttcc 2280 tttetegcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 2340 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca 2400 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 2460 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct 2520 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 2580 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc 2640 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 2700 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 2760 tccgggagct gcatgtgtca gaggttttca ccgtcatcac Cgaaacgcgc gagacgaaag 2820 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 2880 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 2940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 3000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 3060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 3120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 3180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 3240 Pagina 68

P318031NL_ST25{9969840} (1) gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 3300 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 3360 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 3420 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 3480 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 3540 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 3600 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 3660 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 3720 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 3780 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 3840 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 3900 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3960 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 4020 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 4080 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 4140 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 4200 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 4260 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 4320 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 4380 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 4440 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 4500 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 4560 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc agggggEgcgg 4620 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 4680 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4740 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 4800 Pagina 69

P318031NL_ST25{9969840} (1) gaggaagcgg aag 4813 <210> 184 <211> 3758 <212> DNA <213> Artificial Sequence <220> <223> pl5 attB(bxb) lox plasmid <400> 184 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg 60 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag 120 ttggggggag gegtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg 180 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata 240 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta 300 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct 360 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt 420 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag 480 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc 540 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt 600 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt 660 ggggeegegg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc 720 ctgcgagege ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg 780 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg 840 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa 900 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc 960 ttteegtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac 1020 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat 1080 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg 1140 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct 1200

Pagina 70

P318031NL_ST25{9969840} (1) cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tcgccaccat 1260 ggccggcttg tcgacgacgg cggtctccgt cgtcaggatc atccggatcc ataacttcgt 1320 atagcataca ttatacgaag ttatcatgat attcggcaag caggcatcga ctagttaatt 1380 aagctagcgc tgcaagaact cttcctcacg ataacttcgt ataggagact ttatacgaag 14409 ttaagcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 1500 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 1560 gcaccgatac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 1620 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 1680 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 1740 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 1800 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 1860 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 1920 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca 1980 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 2040 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 2100 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 2160 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 2220 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa 2280 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 2340 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 2400 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 2460 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 2520 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 2580 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 2640 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 2700 cgtcagaccc cttaataaga tgatcttctt gagatcgttt tggtctgcgc gtaatctctt 2760 Pagina 71

P318031NL_ST25{9969840} (1) gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc gaaggttctc tgagctacca 2820 actctttgaa ccgaggtaac tggcttggag gagcgcagtc accaaaactt gtcctttcag 2880 tttagcctta accggcgcat gacttcaaga ctaactcctc taaatcaatt accagtggct 2940 gctgccagtg gtgcttttgc atgtctttcc gggttggact caagacgata gttaccggat 3000 aaggcgcagc ggtcggactg aacggggggt tcgtgcatac agtccagctt ggagcgaact 3060 gcctacccgg aactgagtgt caggcgtgga atgagacaaa cgcggccata acagcggaat 3120 gacaccggta aaccgaaagg caggaacagg agagcgcacg agggagccgc Cagggggaaa 3180 cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc 3240 gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc tttgccgcgg ccctctcact 3300 tccctgttaa gtatcttcct ggcatcttcc aggaaatctc cgccccgttc gtaagccatt 3360 tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag tgagcgagga agcggaatat 3420 atcctgtatc acatattctg ctgacgcacc ggtgcagcct tttttctcct gccacatgaa 3480 gcacttcact gacaccctca tcagtgccaa catagtaagc cagtatacac tccgctagcg 3540 ctgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta 3600 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 3660 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 3720 ggcctaggct tttgcaaaaa gctttgcaaa gatggata 3758 <210> 185 <211> 6076 <212> DNA <213> Artificial Sequence <220> <223> pBR_attB(bxb) ccdB lox plasmid <400> 185 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata 60 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta 120 atgtactggg gttgaacact ctgtgccatt ggctccggtg cccgtcagtg ggcagagcgc 180 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag 240

Pagina 72

P318031NL_ST25{9969840} (1) agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc 300 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac 360 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt 420 acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt 480 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc 540 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc 600 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt 660 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat 720 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag 780 cgcacatgtt CggCgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag 840 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc 900 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc 960 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggegggtgag 1020 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg 1080 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct 1140 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact 1200 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt 1260 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag 1320 gtgtcgtgag gaattcgcca ccatggccgg cttgtcgacg acggcggtct ccgtcgtcag 1380 gatcatccgg atccataact tcgtatagca tacattatac gaagttatca tgatattcgg 14409 caagcaggca tcgtagctgc ctagtcttca cacataacca ggaggtcaga ttatgcagtt 1500 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga 1560 tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc 1620 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat 1680 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct 1740 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataacc 1800 Pagina 73

P318031NL_ST25{9969840} (1) cagcccgcct aatgagcggg cttttttttg aacaaaacaa cttatatcgt atggggctga 1860 cttcagaaga catgggagga gctgcaagaa ctcttcctca cgataacttc gtataggaga 1920 ctttatacga agttaagcgc tcactggccg tcgttttaca gggacacgaa gtgatccgtt 1980 taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta 2040 ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg 2100 tgccagccgc ctttgcgacg ctcaccgggc tggttgccct cgccgctggg CLggcggccg 2160 tctatggccc tgcaaacgcg ccagaaacgc cgtcgaagcc gtgtgcgaga caccgeggec 2220 gccggcgttg tggatacctc gcggaaaact tggccctcac tgacagatga ggggcggacg 2280 ttgacacttg aggggccgac tcacccggcg cggcgttgac agatgagggg caggctcgat 2340 ttcggccggc gacgtggagc tggccagcct cgcaaatcgg cgaaaacgcc tgattttacg 2400 cgagtttccc acagatgatg tggacaagcc tggggataag tgccctgcgg tattgacact 2460 tgaggggcgc gactactgac agatgagggg cgcgatcctt gacacttgag gggcagagtg 2520 ctgacagatg aggggcgcac ctattgacat ttgaggggct gtccacaggc agaaaatcca 2580 gcatttgcaa gggtttccgc ccgttttteg gccaccgcta acctgtcttt taacctgctt 2640 ttaaaccaat atttataaac cttgttttta accagggctg cgccctgtgc gcgtgaccgc 2700 gcacgccgaa ggggggtgcc cccccttctc gaaccctccc ggcccgctaa cgcgggcctc 2760 ccatcccccc aggggctgcg cccctcggcc gcgaacggcc tcaccccaaa aatggcagcg 2820 ctggccaatt cccgagtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 2880 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 2940 agtatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 3000 gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 3060 aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 3120 gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 3180 gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 3240 gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 3300 aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 3360 Pagina 74

P318031NL_ST25{9969840} (1) ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 3420 gaagaggaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 3480 cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 3540 gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 3600 gacattgcct tctgcgtccg gtcgctcagg gaggatatcg gggaagaaca gtatgtcgag 3660 ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 3720 ctggatgaat tgttttagct gtcagaccaa gtttactcat atatacttta gattgattta 3780 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 3840 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 3900 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 3960 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 4020 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 4080 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 4140 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 4200 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 4260 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 4320 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 4380 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 4440 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 4500 gccagcaacg cggccttttt acggttcctg gcagatccta gatgtggcgc aacgatgccg 4560 gcgacaagca ggagcgcacc gacttcttcc gcatcaagtg ttttggctct caggccgagg 4620 cccacggcaa gtatttgggc aaggggtcgc tggtattcgt gcagggcaag attcggaata 4680 ccaagtacga gaaggacggc cagacggtct acgggaccga cttcattgcc gataaggtgg 4740 attatctgga caccaaggca ccaggcgggt caaatcagga ataagggcac attgccccgg 4800 cgtgagtcgg ggcaatcccg caaggagggt gaatgaatcg gacgtttgac cggaaggcat 4860 acaggcaaga actgatcgac gcggggtttt ccgccgagga tgccgaaacc atcgcaagcc 4920 Pagina 75

P318031NL_ST25{9969840} (1) gcaccgtcat gcgtgcgccc cgcgaaacct tccagtccgt cggctcgatg gtccagcaag 4980 ctacggccaa gatcgagcgc gacagcgtgc aactggctcc ccctgccctg cccgcgccat 5040 cggccgccgt ggagcgttcg cgtcgtcttg aacaggaggc ggcaggtttg gcgaagtcga 5100 tgaccatcga cacgcgagga actatgacga ccaagaagcg aaaaaccgcc ggcgaggacc 5160 tggcaaaaca ggtcagcgag gccaagcagg ccgcgttgct gaaacacacg aagcagcaga 5220 tcaaggaaat gcagctttcc ttgttcgata ttgcgccgtg gccggacacg atgcgagcga 5280 tgccaaacga cacggcccgc tctgccctgt tcaccacgcg caacaagaaa atcccgcgcg 5340 aggcgctgca aaacaaggtc attttccacg tcaacaagga cgtgaagatc acctacaccg 5400 gcgtcgagct gcgggccgac gatgacgaac tggtgtggca gcaggtgttg gagtacgcga 5460 agcgcacccc tatcggcgag ccgatcacct tcacgttcta cgagctttgc caggacctgg 5520 gctggtcgat caatggccgg tattacacga aggccgagga atgcctgtcg cgcctacagg 5580 cgacggcgat gggcttcacg tccgaccgcg ttgggcacct ggaatcggtg tcgctgctgc 5640 accgcttccg cgtcctggac cgtggcaaga aaacgtcccg ttgccaggtc ctgatcgacg 5700 aggaaatcgt cgtgctgttt gctggcgacc actacacgaa attcatatgg gagaagtacc 5760 gcaagctgtc gccgacggcc cgacggatgt tcgactattt cagctcgcac cgggagccgt 5820 acccgctcaa gctggaaacc ttccgcctca tgtgcggatc ggattccacc cgcgtgaaga 5880 agtggcgcga gcaggtcggc gaagcctgcg aagagttgcg aggcagcggc ctggtggaac 5940 acgcctgggt caatgatgac ctggtgcatt gcaaacgcta gggccttgtg gggtcagttc 6000 cggctggggg ttcagcagcc cctgctcgga tctgttggac cggacagtag tcatggttga 6060 tgggctgcct gtatcg 6076 <210> 186 <211> 4905 <212> DNA <213> Artificial Sequence <220> <223> pBR_attB(C31) FRT plasmid <400> 186 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60

Pagina 76

P318031NL_ST25{9969840} (1) acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta 240 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 300 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 360 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 420 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc 480 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct 540 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 600 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggegeg 660 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 720 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 780 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc 840 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc 900 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg 960 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc 1020 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg 1080 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta 1140 ttteggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg 1200 CgaggCgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc 1260 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gCggcaaggc 1320 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag 1380 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa 14409 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag Laccgggcgc 1500 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggges 1560 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag 1620 Pagina 77

P318031NL_ST25{9969840} (1) cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca 1680 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa 1740 ttcgccacca tggcctcgaa gccgcggtgc gggtgccagg gcgtgccctt gggctccccg 1800 ggcgcgtact ccacctcacc catcggatcc gaagttccta ttccgaagtt cctattctct 1860 agaaagtata ggaacttcat gatattcggc aagcaggcat cgactagtta attaagctag 1920 cgctgcaaga actcttcctc acggatccga agttcctatt ccgaagttcc tattcttcaa 1980 atagtatagg aacttcagcg ctcactggcc gtcgttttac aaagggcgaa ttcactggcc 2040 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 2100 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 2160 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat 2220 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 2280 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2340 tagcgcccgc tcctttegct ttcttecctt cctttctcgc cacgttcgcc ggctttcccc 2400 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2460 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2520 tttttegccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 2580 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 2640 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 2700 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 2760 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 2820 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 2880 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 2940 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 3000 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3060 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 3120 tcegtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 3180 Pagina 78

P318031NL_ST25{9969840} (1) aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 3240 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3300 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 3360 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3420 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 3480 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3540 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3600 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 3660 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 3720 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3780 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 3840 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3900 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 3960 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 4020 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 4080 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4140 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4200 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4260 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 4320 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4380 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4440 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 4500 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 4560 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4620 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 4680 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4740 Pagina 79

P318031NL_ST25{9969840} (1) cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 4800 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 4860 gccgaacgac cgagcgcagc gagtcagtga gCgaggaagc ggaag 4905 <210> 187 <211> 2100 <212> DNA <213> Artificial Sequence <220> <223> Sequence of DNA payload (2 kb) <400> 187 gctggagacc cggctgagtg cagacatggc cactgtcctg cagctgctac agaggcagat 60 gacgctggtc ccgcccgcct acagtgctgt gaccaccccg gggcctggcc ccacttccac 120 atccccgctg ttgcccgtca gccccctccc caccctcacc ttggactcgc tttctcaggt 180 aagctccagc cctgcttcct cgatgtaccg tgacccgctt gttccccgat tccagcccaa 240 acctgctttg ctgcctttct gctgggctct gtggcccagc ctccctgaac cttgttcctg 300 ccctcctccc acccctgtcc tcctggagga aggctgcttc aggagctgca ggctcagggt 360 agggcaggag agtcccccgt ccaaagcccc tagacagctg ggtgcaggga acctgtaggc 420 taagtggccc gcagcttcac tcccctagtg tccacagcct ccaccgeccag gCCCCggagc 480 atcgcctgaa gagccagctc caacggtggt gcctgtctca cggccacggg tggtctccaa 540 gaactgactg agcccgagac gtggcggtgg aggctgcata ccagagtatc tagggacagg 600 gtcccgggtc ccttctgtga gtgacactcc ctggtctcac cgtcctgccc atccttgccc 660 ttggcagtgg ctgtcacagg gtcatcttga gggtgcttgg catttcccag ggtgcagagg 720 ccaagcctcc aggggaggac ttcctgggcc tgctttgccc cctctgctag agatgggcct 780 tagttgcccc ttgtcctctc ctgggtggac ctgcatgttc ctggcctctg caggatggcg 840 gtgtctgtgt caacctccgc gtccatggtg gggactcctc tggctgccag aggcggcctg 900 ggatctgctc cctccccact tcccttggca tccgctcccc cctcccatat gctgcctgct 960 cctgtttgca gacacgaggg cccacgtcag caaatgtgcc agtctccctt cgcagccgcc 1020 cccgcagagc tctcaccagc agacgctcag tacccacaaa cccctcccgc ctgccccggc 1080

Pagina 860

P318031NL_ST25{9969840} (1) cctctccctg gccttcagat gcctggccgc cagcctgcct ctgtttctag gtccccgagt 1140 tctttctgga ttgcgggaag gaatcttgtc tctttttctg tcccagcttc tgtcccaaca 1200 cagagactgc ttgttccagg ttgggttggg ctcagcggcc acattccctc gggaagagac 1260 aggggcctag ggttttccct gctcccagct ctcttgtttg gctcctgctc tcagagaatg 1320 cctttctcta cttcccacct tggtgcctcc ccttggtggc ccctgtcctg ctctcgctgc 1380 cttggctctt gtcccctcca gcttctctgc ccacctgact ctcctcttgg ccctcctgtc 14409 ctcccgtcca tcctctgtcc ccgggttctc ctgccccttt ccctcccctt cctcctccte 1500 catggcctct tcgcctgccc atgctctgtg tgtattgcag gtttcccagt tcatggcgtg 1560 tgaggagctg CCCCCggggg ccccagagct tccccaagaa ggccccacac gacgcctctc 1620 cctaccgggc cagctggggg ccctcacctc ccagcccctg cacagacacg gctcggaccc 1680 gggcagttag tggggctgcc cagtgtggac acgtggctca cccagggatc aaggcgctgc 1740 tgggccgctc cccttggagg ccctgctcag gaggccctga ccgtggaagg ggagaggaac 1800 tcgaaagcac agctcctccc ccagcccttg ggaccatctt ctcctgcagt cccctgggcc 1860 ccagtgagag gggcaggggc agggccggca gtaggtgggg cctgtggtcc ccccactgcc 1920 ctgagggcat tagctggtct aactgcccgg aggcacccgg ccctgggcct taggcacctc 1980 aaggactttt ctgctattta ctgctcttat tgttaaggat aataattaag gatcatatga 2040 ataattaatg aagatgctga tgactatgaa taataaataa ttatcctgag gagaactcca 2100 <210> 188 <211> 5466 <212> DNA <213> Artificial Sequence <220> <223> Sequence of DNA payload (5 kb) <400> 188 gtgctgaagg gcttccctga gtgcctgcag gctgacatct gcctgcacct gaaccgctca 60 ctgctgcagc actgcaaacc cttccgaggg gccaccaagg gctgccttcg ggccctggcc 120 atgaagttca agaccacaca tgcaccgcca ggggacacac tggtgcatgc tggggacctg 180 ctcaccgccc tgtacttcat ctcccggggc tccatcgaga tcctgcgggg cgacgtcgtc 240

Pagina 81

P318031NL_ST25{9969840} (1) gtggccatcc tgggtatggg gtggggggcg ggcactggac tggaaatgcc ctctgcagcc 300 tcaagaggtg cgagccttct gaatatgcag tcactggggc tgtggacctg ggactgcctg 360 cagggtcact gggctccttt aattcaccta aactcaggcc ctccaagcgg ggccatggag 420 aggagcccca cgtggggtga ggctgctgaa ctctggggtt cccacattct ccttccctte 480 aggatccgcc acaaacagac actttttgct tccttaaagt aggatcaaat ctagatcctc 540 tagcctgggc agtagaggaa gaaatgctag cctggaagct cggcatttgg tttcactaag 600 ggccatgtgg ttccctgcag cctcatgcct ggccccttga cacatccaaa gcaaagggag 660 tectgccccc tccccccact tcctttctac cctgcctgtg cacagtgggt gggttggtgt 720 gtctggacac tgaggacttc ctcccccttt gcctgtectt ccctecggccc tgtgtgcctc 780 agggcagata tagcaagctc tttcgaccat agttgatggt aggacatttt agactttgtt 840 tctcagctct gtacaaacac aaatacacac ccccacaaaa ctaaaatcaa agtttcacta 900 cataacactg ggccttactg catgtggttc attctagcat ttctgttctg tgctgtgcta 960 agctatacta ctgtatgttc tttcagtaaa aaaaaaaaaa aaaaaaaaaa aatgctggtt 1020 ttgattcact actgtgttct gatctttggt ttgaagaaca ttgcttataa gggtgcagtg 1080 attggctaag agggtgtttg ggacctgggg tttggggtag tcctgctggc tgggtctcat 1140 ttgctgggag accctgggct cctggcccct ctggcactgt cgtgcctgct gggagctgag 1200 geagggtggg ctggcagatt gcttccccgg ttgtgtgggt Lgggggccgg ctggeaggeg 1260 acccagctga ggggacatgc tctgcccggg gtggggatgc cgggagaagg tgcctgctgc 1320 ctggatgccg ccccccgggg ctgagctccc tgtcctctcc atggcctcca gggaagaatg 1380 acatctttgg ggagcctctg aacctgtatg caaggcctgg caagtcgaac ggggatgtgc 14409 gggccctcac ctactgtgac ctacacaaga tccatcggga cgacctgctg gaggtgctgg 1500 acatgtaccc tgagttctcc gaccacttct ggtccagcct ggagatcacc ttcaacctgc 1560 gagatgtgag ttggctgccc tgagctggac cccatcctcc tgcttccagc tgtgtggctg 1620 cccctttgct gtgtgacatt gaatgtgatg cttctctgtc tcagtttccc tgtctgtcaa 1680 atggtatcat agagcagcct acaaaagtcc caaactcagt ctttgcctgg ggcaaaatca 1740 cattgggggc agggaagggt tttcccagag ctggggcgcc cagccctact ttttctgtgt 1800 Pagina 82

P318031NL_ST25{9969840} (1) taaggaggga gcttggggcc tgaccccggt ggggcaggag agcactgaaa gggccctgat 1860 actgattttg gttccagacc aacatgatcc cgggctcccc cggcagtacg gagttagagg 1920 gtggcttcag tcggcaacgc aagcgcaagt tgtccttccg caggcgcacg gacaagggtg 1980 aggcgegeea ggggaggaag ggegagegscg gggacaaggt gaggctggga gctggaaggc 2040 geggtgggcc gtgtctgctt tacccaggcc tcagaccttc ccatcccttc ctccagtgtg 2100 cccactccac accccagctg tccagatgct gcccatcttt cagggcccct gcagctgtca 2160 cctcctccag gaagccctct tccaccctcc agggcaagcc taggcttttt ggacttcaca 2220 gegctggggt ctctgtgagc actgaggagg aaagaacata gaacaaagga gggccaggga 2280 gccaacgatg gggctgaggc cccgcagtcc cagagatccg ccaacccctg aagcaccatt 2340 gccagtgctt gggcagtcag ggggtcctgg gagttcttgg tcggcttgac agtgggtgca 2400 agatagcaga agaagcgact ggggacaaac cagggcttcc tgcccagtcc tctctcgggc 2460 tegctcctgt cccctcctcc accctegccec cctectetet gttetcctcc cctctctgag 2520 gcccattctc tgtttcccac agacacggag cagccagggg aggtgtcggc cttggggccg 2580 ggcegggegg gggreagggec gagtagccgg SECCggCCgg gggggecgtyg GEEZEagagC 2640 ccgtccagtg gcccctccag ccctgagagc agtgaggatg agggcccagg ccgcagctcc 2700 agccccctcc gcctggtgcc cttctccagc cccaggcccc ccggagagcc geegggtges 2760 gagcccctga tggaggactg cgagaagagc agcgacactt gcaaccccct gtcaggtatc 2820 CCggECgaCg gECggECgag ggaggaccgg gcgtggcagc ggtggtgcgt ctaccccgct 2880 cacccagctc tgctctctgg ctgcaggcgc cttctcagga gtgtccaaca ttttcagctt 2940 ctggggggac agtcggggcc gccagtacca ggagctccct cgatgccccg cccccacccc 3000 cagcctcctc aacatccccc tctccagccc gggteggegg CCCCggggcg acgtggagag 3060 caggctggat gccctccagc gccagctcaa caggtgaggg agtgcaggtg gggtggeges 3120 gcacgccctg gagtctggtc caggtcctgg cggtgttgtc tggtagaggg agagggcctg 3180 tctgtgtgtg cagcagctgg atccccttct tccattccta gcccagctgg gggctgccac 3240 acctctagcg cggagggcct gggttgacag gctgcccctc ccccctcccc ttccccttcc 3300 ccactcccgg tggaggctgt cactggtgtc cccacttctc tgagcatccc ccacttcctg 3360 Pagina 83

P318031NL_ST25{9969840} (1) ccccaggctg gagacccggc tgagtgcaga catggccact gtcctgcagc tgctacagag 3420 gcagatgacg ctggtcccgc ccgcctacag tgctgtgacc accccggggc ctggccccac 3480 ttccacatcc ccgctgttgc ccgtcagccc cctccccacc ctcaccttgg actcgctttc 3540 tcaggtaagc tccagccctg cttcctcgat gtaccgtgac ccgcttgttc cccgattcca 3600 gcccaaacct gctttgctgc ctttctgctg ggctctgtgg cccagcctcc ctgaaccttg 3660 ttectgccct cctcccaccc ctgtcctcct ggaggaaggc tgcttcagga gctgcaggct 3720 cagggtaggg caggagagtc ccccgtccaa agcccctaga cagctgggtg cagggaacct 3780 gtaggctaag tggcccgcag cttcactccc ctagtgtcca cagcctccac cgccaggccc 3840 cggagcatcg cctgaagagc cagctccaac ggtggtgcct gtctcacggc cacgggtggt 3900 ctccaagaac tgactgagcc cgagacgtgg cggtggaggc tgcataccag agtatctagg 3960 gacagggtcc cgggtccctt ctgtgagtga cactccctgg tctcaccgtc ctgcccatcc 4020 ttgcccttgg cagtggctgt cacagggtca tcttgagggt gcttggcatt tcccagggtg 4080 cagaggccaa gcctccaggg gaggacttcc tgggcctgct ttgccccctc tgctagagat 4140 gggccttagt tgccccttgt cctctcctgg gtggacctgc atgttcctgg cctctgcagg 4200 atggcggtgt ctgtgtcaac ctccgcgtcc atggtgggga ctcctctggc tgccagaggc 4260 ggcctgggat ctgctccctc cccacttccc ttggcatccg ctcccccctc ccatatgctg 4320 cctgctcctg tttgcagaca cgagggccca cgtcagcaaa tgtgccagtc tcccttcgca 4380 gccgcccccg cagagctctc accagcagac gctcagtacc cacaaacccc tcccgcctgc 4440 cccggccctc tccctggcct tcagatgcct ggccgccagc ctgcctctgt ttctaggtcc 4500 ccgagttctt tctggattgc gggaaggaat cttgtctctt tttctgtccc agcttctgtc 4560 ccaacacaga gactgcttgt tccaggttgg gttgggctca gcggccacat tccctcggga 4620 agagacaggg gcctagggtt ttccctgctc ccagctctct tgtttggctc ctgctctcag 4680 agaatgcctt tctctacttc ccaccttggt gcctcccectt ggtggcccct gtcctgctct 4740 cgctgccttg gctcttgtcc cctccagctt ctctgcccac ctgactctcc tcttggccct 4800 cctgtcctcc cgtccatcct ctgtcccegg gttctcctgec cccetttccct ccccttccte 4860 ctcctccatg gcctcttcgc ctgcccatgc tctgtgtgta ttgcaggttt cccagttcat 4920 Pagina 84

P318031NL_ST25{9969840} (1) ggcgtgtgag gagctgcccc cCgggggcccc agagcttccc Caagaaggcc ccacacgacg 4980 cctctcccta ccgggccagc tgggggccct cacctcccag cccctgcaca gacacggctc 5040 ggacccgggc agttagtggg gctgcccagt gtggacacgt ggctcaccca gggatcaagg 5100 cgctgctggg ccgctcccct tggaggccct gctcaggagg ccctgaccgt ggaaggggag 5160 aggaactcga aagcacagct cctcccccag cccttgggac catcttctcc tgcagtcccc 5220 tgggccccag tgagagggegc aggggcaggg ccggcagtag gtggggcctg tggtcccccc 5280 actgccctga gggcattagc tggtctaact gcccggaggc acccggccct gggccttagg 5340 cacctcaagg acttttctgc tatttactgc tcttattgtt aaggataata attaaggatc 5400 atatgaataa ttaatgaaga tgctgatgac tatgaataat aaataattat cctgaggaga 5460 actcca 5466 <210> 189 <211> 10549 <212> DNA <213> Artificial Sequence <220> <223> Sequence of DNA payload (10 kb) <400> 189 atggcggeccc CagCCgggaa ggCgagcagg acaggggctc LgCggcccag ggcccagaaa 60 ggccgggtga ggcgggccgt gcgcatctcc agcctcgtgg cccaggaggt aggtgacacc 120 cggtccttcc tcttctttct ggggccggca cactgaggca ggtggctggc ccaagggctc 180 tgactccagg ctggggagag ggctgctggg aagtaggaag ctttgggagc cgtggggcct 240 ggccctgctc acccgcgcct ggctcctgtt ctcattccct cccaggctgg gcttacagag 300 tttccgttge tgggacttcc ccgctcctgt gcctgccatg gcccacagtc ctggagctca 360 aaatgcaaag tggggagatg acaggaatga gatgggtgcc cagagagatg gtggggaggc 420 ggagtgggac aaaggagccc atgggcagga ccaggagaag CCggCaggcg acagctgggc 480 ctggcatgga ggcgccgggg ctcctttgct ctgtgtgctg gggttggacc ctgtgggcaa 540 aggaggcttg tgctgttttg gggggtcgag gaggctggaa tgtgcggaag ccactgtgtg 600 ggtggtgagg gcgcgctggg ctgaggcacc tggctcatct cttcatgggc agccgaggtg 660

Pagina 85

P318031NL_ST25{9969840} (1) tggtggctgg gccatgcact actctgtgac ctcctgggag ccatccagcc gctcttctgt 720 cagcctgggg caggcttggg ccgacagtgg aggaggagtg ggaggtggct cagggctaag 780 tctgggaatc ccatccctgc ccccaccacc ccacatctgc acattctgcc ttggctcata 840 cactctttcc agctggggga caggtacagg taggtcttcc cacccacagc tgaggagtca 900 gegtttggct taagcaagcc agcttcagta ttctatagga caacaggctg gggggacaga 960 cctggcccaa ggccataggt gagctggtgg ctgcagagct gggactggcc cctgtcaggt 1020 gtgccccatg cacgtgtgag tttgtaccct ggggcagtgt tgctcatgct gtgtgcagac 1080 tcaggggtag ggtcactgct gttaggggtg gtcctgacac ctccgcccag aggcttgacc 1140 tctcaggcac agagaagagc cctgtccagg ggcagctacc ttcttctgcc cccttatcag 1200 caggggcctg gccagtgttt tctttttttt ttttcagtcc ccacttgatc actgaagggc 1260 cacccaccca tgggcatttg ctctctgcct taaggagctg ggcacttaga cagcagacag 1320 agcagaaatg gagcctgtga gcctgtccat gtgttccctc cctgccctag gcctcagctg 1380 ctcccgcagc ccatctggta ccgctggagg ccctgtgtcc accccagggt ggtcaaatct 14409 ttgactatga cccgtcttga cttctagtta tttcctctaa ggagatgttc tcccagcagc 1500 gccatctgtg aaggtgtctt gggcaagccc agcttgagta tgaaggggag tctttgggac 1560 tcatcaacta tggcctcccc aacccaaaag cctgagggtg tgggggcctg ggagtcagtg 1620 cttctgggag cagggccagc acctctggaa ttcatggggg ctgggcaagg ccttgggttc 1680 agctggctgc ccagggatgg cagggagagc ttggggaggg ggcttttggg ttgagctgca 1740 tgcctgagag aggattctag ggagacccag aagcttctgg gacagtgtgg agaagctggc 1800 cggagctctt cttagtttgc cttgggagca agagggacgt gcctgctgtc accgggtccg 1860 gatggccagc ctgcctctgt ctgatgactg tggaggaggc agagtgtgag tgtgagctgg 1920 tacaggggct gtgtgtgtca ctgctgagca gtccctgtgt gcacgtccct ctgtataagt 1980 tgggtgccat ctcctcccag ctgggctatg tcttctttgt ccctttgtaa gagggtggga 2040 ggatgaggac tgtgggctgt cccggacctg tgtggagagg gggctgaggg agcgaggtgt 2100 gcgagtggag ggtgggagtg tcttggtctt tctccatcct ggttgcctgg tgtcctcggg 2160 cctgtggcat gaagaggggc tggtaagaac tgcagtgact accagtcagt ggattggggc 2220 Pagina 86

P318031NL_ST25{9969840} (1) tatggctcct ggggccactg cccaggttgg gagctgttgt ccttggaacc ctttccggtc 2280 tgagactatg gcgtgtgtgg ttgggtgtgt gggccagcgt ctcagggcag tcacagtggc 2340 aggtctgcat ctgcattgcc ggcggggagc agcactgggt gttcctgccg agtggcctga 2400 atccacgtct gccgggaaat gttggaactg tttggagcca gtcctgtgct gtgtggcatt 2460 ttgggaacca tgatgcccaa acctgggggt ggccatttgc tgtggtcgct tggctgaggg 2520 gtgggcattc tgatggaagc tttttttggc ctcctcctca ttctgcttgg ccccggcaga 2580 gatgtcatcg ctcctgcccc cgaggtccca tggcctgcct cacccacacc tccgccttcc 2640 ccgggtgcag gtcctgtccc tgggcgccga cgtgctgcct gagtacaagc tgcaggcacc 2700 gcgcatccac cgctggacca tcctgcatta cagccccttc aaggccgtgt gggactggct 2760 catcctgctg ctggtcatct acacggctgt cttcacaccc tactcggctg ccttcctgct 2820 gaaggagacg gaagaaggcc cgcctgctac cgagtgtggc tacgcctgcc agccgctggc 2880 tgtggtggac ctcatcgtgg acatcatgtt cattgtggac atcctcatca acttccgcac 2940 cacctacgtc aatgccaacg aggaggtggt cagccacccc ggccgcatcg ccgtccacta 3000 cttcaagggc tggttcctca tcgacatggt ggccgccatc cccttcgacc tgctcatctt 3060 cggctctggc tctgaggagg tggggtcagc aaggaggcag gtggtgtagg gagaggagag 3120 gccaggaatg tggtgggagg tagtggggtg gagggggtegg cgaggaggac agagaaaaag 3180 agagagagag agaaagagag aaagacagtg ggagagagac atggtggcaa ggcagctgca 3240 gaggcaaggt gggtacccca gcaaacctat gctccttctc tccacacaag ctcctcgcag 3300 gttgcagagg gccacacagg acaccttttt ttctaattcg tccatcccaa ggggccatta 3360 ttttgtagcg tcaccaaagt gccctagaag ctggtgggag tgggcaggca ccctagggtg 3420 atcactgcac ctgtcagtgc ccctgtttcc tcatccacac tatgggggct gcctctgtcc 3480 caaagctagc actaagatta atgagacacc tgtgctgggc ccagtgtggg cttcacctct 3540 taggaggagg gtctaggaag tctttgggga tctgacctct gatgctcgct ctgagggccg 3600 cagcacctcc cctcctccct tgccccecctt gccccatcaa cggaatgtgc cccttccctg 3660 tcccccagct gatcgggctg ctgaagactg cgcggctgct gcggctggtg CgCgtggcgc 3720 ggaagctgga tcgctactca gagtacggcg cggccgtgct gttcttgctc atgtgcacct 3780 Pagina 87

P318031NL_ST25{9969840} (1) ttgcgctcat cgcgcactgg ctagcctgca tctggtacgc catcggcaac atggagcagc 3840 cacacatgga ctcacgcatc ggctggctgc acaacctggg cgaccagata ggcaaaccct 3900 acaacagcag cggcctgggc ggcccctcca tcaaggacaa gtatgtgacg gcgctctact 3960 tcaccttcag cagcctcacc agtgtgggct tcggcaacgt ctctcccaac accaactcag 4020 agaagatctt ctccatctgc gtcatgctca ttggctgtga gtgtgcccag gggCggECEgEg 4080 Cggggagagc ccacggtgga ggaacccaag ttggaggaaa ctgaggctgc tagccgggcc 4140 agagtgaggc cctggaactt agagactcca ggggccacca tcgtgacatg gtttgggggc 4200 tgggtcacca gggcggggac tactgaaggg tggggtgtgc tggagcgcag atgtacaagg 4260 gcgtctcgtg ggcatgtcga cgctgagact gagacactga cctggtgcgg ggcctgagca 4320 gegtccctgt gggtgggtgg ggtccctgca gaggctgacg gccccacgtg cccacgcccc 4380 cagccctcat gtatgctagc atcttcggca acgtgtcggc catcatccag cggctgtact 4440 cgggcacagc ccgctaccac acacagatgc tgcgggtgcg ggagttcatc cgcttccacc 4500 agatccccaa tcccctgcgc cagcgcctcg aggagtactt ccagcacgcc tggtcctaca 4560 ccaacggcat cgacatgaac gcggtgaggc caccagagcg tggccagtgg gtggcaggct 4620 gggaagagtg gggtggcaga ggggaacctc ttggcacagc aaacaagtct caggcggtca 4680 cagtcagtag taaggaccct ggaggcacgt tgcccctttt tgcccatgag gaaatggaag 4740 cccttggaac cccttccctg gagagctccc cttttggggt gggaggtata tgtcacatag 4800 gcgatggggt taaatccttg tgacagccca gggcgggaat ggtctctatg ccttacaaat 4860 aagaacatgg aggtctagag cccaagggtg ctcggccagg catggtgaag ctggagtggg 4920 gtcccaggtc cttcccaaga cactgggccc cctgatgctt ccgagatctc ccaggcctgg 4980 aggttgagat ttctctgaca tggaggggtg ggatggtgga gtagagtgtg ggttgggges 5040 tcccaaggga gggtgtgctg agctgccccc accctgcccc caggtgctga agggcttccc 5100 tgagtgcctg caggctgaca tctgcctgca cctgaaccgc tcactgctgc agcactgcaa 5160 acccttccga ggggccacca agggctgcct tcgggccctg gccatgaagt tcaagaccac 5220 acatgcaccg ccaggggaca cactggtgca tgctggggac ctgctcaccg ccctgtactt 5280 catctcccgg ggctccatcg agatcctgcg gggcgacgtc gtcgtggcca tcctgggtat 5340 Pagina 88

P318031NL_ST25{9969840} (1) ggggtggggg gcgggcactg gactggaaat gccctctgca gcctcaagag gtgcgagcct 5400 tctgaatatg cagtcactgg ggctgtggac ctgggactgc ctgcagggtc actgggctcc 5460 tttaattcac ctaaactcag gccctccaag cggggccatg gagaggagcc ccacgtggge 5520 tgaggctgct gaactctggg gttcccacat tctccttccc ttcaggatcc gccacaaaca 5580 gacacttttt gcttccttaa agtaggatca aatctagatc ctctagcctg ggcagtagag 5640 gaagaaatgc tagcctggaa gctcggcatt tggtttcact aagggccatg tggttccctg 5700 cagcctcatg cctggcccct tgacacatcc aaagcaaagg gagtcctgcc ccctcccccc 5760 acttcctttc taccctgcct gtgcacagtg ggtgggttgg tgtgtctgga cactgaggac 5820 ttectccccc tttgcctgtc cttccctcgg ccctgtgtgc ctcagggcag atatagcaag 5880 ctctttcgac catagttgat ggtaggacat tttagacttt gtttctcagc tctgtacaaa 5940 cacaaataca cacccccaca aaactaaaat caaagtttca ctacataaca ctgggcctta 6000 ctgcatgtgg ttcattctag catttctgtt ctgtgctgtg ctaagctata ctactgtatg 6060 ttctttcagt aaaaaaaaaa aaaaaaaaaa aaaaatgctg gttttgattc actactgtgt 6120 tctgatcttt ggtttgaaga acattgctta taagggtgca gtgattggct aagagggtgt 6180 ttgggacctg gggtttgggg tagtcctgct ggctgggtct catttgctgg gagaccctgg 6240 gctcctggcc cctctggcac tgtcgtgcct gctgggagct gagggagggt gggctggcag 6300 attgcttccc cggttgtgtg ggttgggggc cggctggcag gcgacccagc tgaggggaca 6360 tgctctgccc ggggtgggga tgccgggaga aggtgcctgc tgcctggatg ccgccccccg 6420 gggctgaget ccctgtcctc tccatggcct ccagggaaga atgacatctt tggggagcct 6480 ctgaacctgt atgcaaggcc tggcaagtcg aacggggatg tgcgggccct cacctactgt 6540 gacctacaca agatccatcg ggacgacctg ctggaggtgc tggacatgta ccctgagttc 6600 tccgaccact tctggtccag cctggagatc accttcaacc tgcgagatgt gagttggctg 6660 ccctgagctg gaccccatcc tcctgcttcc agctgtgtgg ctgccccttt gctgtgtgac 6720 attgaatgtg atgcttctct gtctcagttt ccctgtctgt caaatggtat catagagcag 6780 cctacaaaag tcccaaactc agtctttgcc tggggcaaaa tcacattggg ggcagggaag 6840 ggttttccca gagctggggc gcccagccct actttttctg tgttaaggag ggagcttggg 6900 Pagina 89

P318031NL_ST25{9969840} (1) gcctgacccc ggtggggcag gagagcactg aaagggccct gatactgatt ttggttccag 6960 accaacatga tcccgggctc ccccggcagt acggagttag agggtggctt cagtcggcaa 7020 cgcaagcgca agttgtcctt CCgCaggcgc acggacaagg gLgaggCgEgg gEagggEgagEg 7080 aagggggagg gcggggacaa ggtgaggctg ggagctggaa ggcggggtgg gccgtgtctg 7140 ctttacccag gcctcagacc ttcccatccc ttcctccagt gtgcccactc cacaccccag 7200 ctgtccagat gctgcccatc tttcagggcc cctgcagctg tcacctcctc caggaagccc 7260 tcttccaccc tccagggcaa gcctaggctt tttggacttc acagggctgg ggtctctgtg 7320 agcactgagg aggaaagaac atagaacaaa ggagggccag ggagccaacg atggggctga 7380 ggccccgcag tcccagagat ccgccaaccc ctgaagcacc attgccagtg cttgggcagt 7440 cagggggtcc tgggagttct tggtcggctt gacagtgggt gcaagatagc agaagaagcg 7500 actggggaca aaccagggct tcctgcccag tcctctcteg ggctcgctcc tgtcccctcc 7560 tccacccteg ccccctcctc tctgttetce tcccctetct gaggcccatt ctctgtttcc 7620 cacagacacg gagcagccag gggaggtgtc ggccttgggg CCggECCgEgg CEggEgECagEg 7680 gccgagtage cggggecgge cgggggggcc gtggggggag agcccgtcca gtggcccctc 7740 cagccctgag agcagtgagg atgagggccc aggccgcagc tccagccccc tccgcctggt 7800 gcccttctcc agccccaggc cccccggaga gccgccgggt ggggagcccc tgatggagga 7860 ctgcgagaag agcagcgaca cttgcaaccc cctgtcaggt atcccgggeg aCgggcgggc 7920 gagggaggac cgggcgtggc agcggtggtg cgtctacccc gctcacccag ctctgctctc 7980 tggctgcagg cgccttctca ggagtgtcca acattttcag cttctggggg gacagtcggg 8040 gccgccagta ccaggagctc cctcgatgcc ccgcccccac ccccagcctc ctcaacatcc 8100 ccctctccag cccgggtcgg cggccccggg gcgacgtgga gagcaggctg gatgccctcc 8160 agcgccagct caacaggtga gggagtgcag gtggggtggg ggggcacgcc ctggagtctg 8220 gtccaggtcc tggcggtgtt gtctggtaga gggagagggc ctgtctgtgt gtgcagcagc 8280 tggatcccct tcttccattc ctagcccagc tgggggctgc cacacctcta gCgCggaggg 8340 cctgggttga caggctgccc ctcccccctc cccttcccct tccccactcc cggtggaggc 8400 tgtcactggt gtccccactt ctctgagcat cccccacttc ctgccccagg ctggagaccc 8460 Pagina 90

P318031NL_ST25{9969840} (1) ggctgagtge agacatggcc actgtcctgc agctgctaca gaggcagatg acgctggtcc 8520 cgcccgccta cagtgctgtg accaccccgg ggcctggccc cacttccaca tccccgctgt 8580 tgcccgtcag ccccctcccc accctcacct tggactcgct ttctcaggta agctccagcc 8640 ctgcttcctc gatgtaccgt gacccgcttg ttccccgatt ccagcccaaa cctgctttgc 8700 tgcctttctg ctgggctctg tggcccagcc tccctgaacc ttgttcctgc cctcctccca 8760 cccctgtcct cctggaggaa ggctgcttca ggagctgcag gctcagggta gggcaggaga 8820 gtcccccgtc caaagcccct agacagctgg gtgcagggaa cctgtaggct aagtggcccg 8880 cagcttcact cccctagtgt ccacagcctc caccgccagg ccccggagca tcgcctgaag 8940 agccagctcc aacggtggtg cctgtctcac ggccacgggt ggtctccaag aactgactga 9000 gcccgagacg tggcggtgga ggctgcatac cagagtatct agggacaggg tcccgggtcc 9060 cttctgtgag tgacactccc tggtctcacc gtcctgccca tccttgccct tggcagtggc 9120 tgtcacaggg tcatcttgag ggtgcttggc atttcccagg gtgcagaggc caagcctcca 9180 ggggaggact tcctgggcct gctttgcccc ctctgctaga gatgggcctt agttgcccct 9240 tgtcctctcc tgggtggacc tgcatgttcc tggcctctgc aggatggcgg tgtctgtgtc 9300 aacctccgcg tccatggtgg ggactcctct ggctgccaga ggcggcctgg gatctgctcc 9360 ctccccactt cccttggcat ccgctccccc ctcccatatg ctgcctgctc ctgtttgcag 9420 acacgagggc ccacgtcagc aaatgtgcca gtctcccttc gcagccgccc ccgcagagct 9480 ctcaccagca gacgctcagt acccacaaac ccctcccgcc tgccccggcc ctctccctgg 9540 ccttcagatg cctggccgcc agcctgcctc tgtttctagg tccccgagtt ctttctggat 9600 tgcgggaagg aatcttgtct ctttttctgt cccagcttct gtcccaacac agagactgct 9660 tgttccaggt tgggttgggc tcagcggcca cattccctcg ggaagagaca ggggcctagg 9720 gttttecctg ctcccagctc tcttgtttgg ctcctgctct cagagaatgc ctttctctac 9780 ttcccacctt ggtgcctccc cttggtggcc cctgtcctgc tctcgctgcc ttggctcttg 9840 tcccctccag cttctctgcc cacctgactc tcctcttggc cctcctgtcc tcccgtccat 9900 cctctgtccc cgggttctcc tgcccctttec cctcccctte ctcctcctcc atggcctctt 9960 cgcctgccca tgctctgtgt gtattgcagg tttcccagtt catggcgtgt gaggagctgc 10020 Pagina 91

P318031NL_ST25{9969840} (1) ccececggggge cccagagctt ccccaagaag gccccacacg acgcctctcc ctaccgggcc 10080 agctgggggc cctcacctcc cagcccctgc acagacacgg ctcggacccg ggcagttagt 10140 geggctgccc agtgtggaca cgtggctcac ccagggatca aggcgctgct gggccgctcc 10200 ccttggaggc cctgctcagg aggccctgac cgtggaaggg gagaggaact cgaaagcaca 10260 gctcctcccc cagcccttgg gaccatcttc tcctgcagtc ccctgggccc cagtgagagg 10320 ggcaggggca gggccggcag taggtggggc ctgtggtccc cccactgccc tgagggcatt 10380 agctggtcta actgcccgga ggcacccggc cctgggcctt aggcacctca aggacttttc 10440 tgctatttac tgctcttatt gttaaggata ataattaagg atcatatgaa taattaatga 10500 agatgctgat gactatgaat aataaataat tatcctgagg agaactcca 10549 <210> 190 <211> 14782 <212> DNA <213> Artificial Sequence <220> <223> Sequence of DNA payload (15 kb) <400> 190 ggagctgctt cctatgtctg gtggatgtgg tgcccgtgaa gaacgaggat ggggctgtca 60 tcatgttcat cctcaatttc gaggtggtga tggagaagga catggtgggg tccccggctc 120 atgacaccaa ccaccggggc ccccccacca gctggctggc cccaggtaag tgtacttgct 180 ttagggcagg ctcaggggtt cgtggtctca tttctttggg gctgtgagct gtccaaggtc 240 aacgctctgc aggaagggag gatgtaatgg ttgggtgggg ggtcccccct ttggaggtgg 300 gaagtgaacc ctgtaccctg aggatgtggc ttggttcccc tggcccagga tggacacata 360 ggggtggaat gacagggccc agaatcacag gtccttggcg tgggctagca ccgtttccct 420 agcttcatct gttgggcttc ccatcccagc atcccctggg agcggtcctc tctccagcct 480 ggcttgagca gcccagggtg gggaacatga ccacctggga gcagctcctc ccctcagtag 540 gaagctgtgt cctatggact cagctgtgtg tagtttcttt catctgctcc ttgttcatgg 600 actggccaag tttcctgggg gtctgggcta gctgaggaga ggctttgagg gaatgcccac 660 ctgggctgga ccatcccctt gacaacaaag gctcggagcc ccttcctctg tcaggatgcc 720

Pagina 92

P318031NL_ST25{9969840} (1) ctctttggct taattttaat ctttccctca gcatcatgac aaaagtgctc acctcttggt 780 ttcttacact ctaaaatgac agtgtcagta aaactggccc tacctgctca tcggatgcca 840 cccctgacgg cagggcccct ggcattgtgt cgcccactta ggcgcaggcc ccacgcactg 900 gctgtgtgac ctccagagag tgacatgacg cctttgtgtc cacacttcct catgtctaaa 960 atggggacgc tggcacccag cggccggtgt gagagacagg gatgatttat ggaatccgcc 1020 ctgaagtgtc acttcagata tggcggttta tgatgactgg attctagaat gatttttgag 1080 actcgaggtc aaaatattgt gaatggaagt ggggatgggg ggttgcccgt tcccctcectt 1140 cccttaccca gcggctgggg gtccatttcc caggccttgc ccgctccggg gctgctcgcg 1200 atccccacga ccacgtgcct ctcctctccc gcaggccgcg ccaagacctt ccgcctgaag 1260 ctgcccgcgc tgctggcgct gacggcccgg gagtcgtcgg LgCggtcggg CggCgCgggC 1320 gECgcgggcg ccccgggggc cgtggtggtg gacgtggacc Lgacgcccgc ggcacccagc 1380 agcgagtcgc tggccctgga cgaagtgaca gccatggaca accacgtggc agggctcggg 14409 cccgcggagg agcggcgtgc gctggtgggt cccggctctc CgCCCCgCag CgCgCCCggC 1500 cagctcccat cgccccgggc gcacagcctc aaccccgacg cctcgggctc cagctgcagc 1560 ctggcccgga cgcgctcccg agaaagctgc gccagcgtgc gccgcgcctc gtcggccgac 1620 gacatcgagg ccatgcgcgc cggggtgctg CCCCCgCcac cgcgccacgc cagcaccggt 1680 gagggcgccg cgggacccgt tccgccccag cccacgcttc ttctgctgcc aggcttgctg 1740 cattctgggc cctcggcttc tgaccttttc actacgtcct gggtgcctgg gagatggaaa 1800 tcccattaag cacttattta attccattaa gtggcttgtt gcgctattaa atgtccattc 1860 atcccattac attcttccac tcccagagcc ctggtgggcc cctggagctg gctgtcccgg 1920 gacggcctgg agcagggatg ccctgggcag gagacgtgcc cagcctccga acaccacctg 1980 gccccctgcc tgccagcgtc ctggagcaga ccgtttcctc ttcctcttcc tggtctcagg 2040 gctgtctcct ggctgcttcc ttagagtggg acaggggtct gacctctctt gtttcttgtg 2100 actcccctgg tctcttgagc tgaccccatg gactcctgac tgtgcagggc ctggtctcca 2160 ctctcgatct atgggtggcc tgcctggcct ggggccctgg gctgctggcc tgaccacgct 2220 gcctctccac ctaggggcca tgcacccact gcgcagcggc ttgctcaact ccacctcgga 2280 Pagina 93

P318031NL_ST25{9969840} (1) ctccgacctc gtgcgctacc gcaccattag caagattccc caaatcaccc tcaactttgt 2340 ggacctcaag ggcgacccct tcttggcttc gcccaccagt gaccgtgaga tcatagcacc 2400 taagataaag gagcgaaccc acaatgtcac tgagaaggtc acccaggtag gcgcccagcg 2460 gccgggctct cctctcacct tggaggagct tggagagggg gtgggagtgg gctgtgatcc 2520 agagccgggt ggagggggct agccagtgag ggcctcttgg agggaggcag acctgagggt 2580 tggggttggc aatctattcc agagctgcga tggggggaga ggggacagtc tgagaagtcc 2640 tggagagggg actggagagg tctggtaaag ggctgggaat ggcagagcgg aggtcgggtg 2700 gatggggtgc gtgccttcgg ggcacagggt gaggacaggg cagttgaggc tggggacatg 2760 acagggccag tagcaagggg ggaaggcggc ctgagggaca tgaaaggtct ggctgcaggg 2820 tgtgtgaagg aagtactagg ggccaagcca actgggaggc ttcaaatgag aaggggtcac 2880 agaggtcaag gacaggaggg tcctaattct tccaggccaa ggtgatgtgc caggcagagg 2940 gtgggcctca gtcctgtggg gatggccagg atggcctcga ggggcccaac tatgtcccta 3000 aagcatggga gaagcaagtt ttcttctcat agggaggtgg tgggtacaaa gatatggaag 3060 ggtcaggtcc ctgaatcctg ggccagagaa gacccctcag ccttgggggc attagccagc 3120 ctggtcttct gtcccctcct gggtttgggg tcctcctggc agcaagagga cctgcgaccc 3180 ctccatggct tcatccaccc gacaccttat cctagccttt ggggectggg ggtgggtges 3240 tcaggagtgc agatctctgc agtgagtgag aagcctggga taatgtggag gactcctgtc 3300 ccaagctgtt ccctcctgcc ccttccacag cagctgcctg ggttggctca tgtaagggag 3360 ctgccctgct acctcggcgg gctcctggcc ctgctcccac cgcctggctt tctgggtccc 3420 tgccaggtct ccagcccact ggccttgact ccagagcatt ccccctcacc ccaggccctt 3480 atctggccac ctacccctct cttcctcctc ccttgettet ctcttggttc ccacctgagc 3540 ccctggcatc tgccctgctc ctgtgcccgc tgcatgtgcc cgtgagttgc ggtgggtctg 3600 tgcatgtgcg cctgtatgtg tcggcaggcg tgtgtgttcc tgccagtcac tagcagtctc 3660 tccatctctg ggtgtctctg tctgactctg gctggctcag cactggaccg gCtgggCgEgg 3720 ggtgtcaggg agaccattaa tctgcggtga caggcccggc Lgctggcgga gEgEggaggEEg 3780 ctcaggctgc gggagcgccg ctgcaggagc CLgggCgggc LgCgggEgag ggggccgctg 3840 Pagina 94

P318031NL_ST25{9969840} (1) gctgggeacc ggCggggcgg gggagcctag tgtggegggg tggggetggg gacctggtag 3900 aaggaggggc cggctggagg agctgaggtt ccgagtgcgg ccgctgctgg gCLggCgggc 3960 gggcagagca cggcaccctg gCagcagggc ccacgccacg gggccatggg cagctcgagc 4020 caggcaggct gctgcccacg cttactgcca gggtgacccc agccctgggg cccagccaca 4080 accaccctgg cttcatgcca ggggctgctc tggttgccag tcggccagcc tcgggggtgc 4140 agcctgggct gggactgctg ctggggtgca ggtgaggcag tggccgggcc ctcaggcccc 4200 agggcaggca ggctgcaggg agccaagtcc tccatggegg CCCCagCCgg gaaggcgagc 4260 aggacagggg ctctgcggcc cagggcccag aaaggccggg tgaggcgggc cgtgcgcatc 4320 tccagcctcg tggcccagga ggtaggtgac acccggtcct tcctcttctt tctggggccg 4380 gcacactgag gcaggtggct ggcccaaggg ctctgactcc aggctgggga gagggctgct 4440 gggaagtagg aagctttggg agccgtgggg cctggccctg ctcacccgcg cctggctcct 4500 gttctcattc cctcccaggc tgggcttaca gagtttccgt tgctgggact tccccgctcc 4560 tgtgcctgcc atggcccaca gtcctggagc tcaaaatgca aagtggggag atgacaggaa 4620 tgagatgggt gcccagagag atggtgggga ggcggagtgg gacaaaggag cccatgggca 4680 ggaccaggag aagccggcag gcgacagctg ggcctggcat ggaggcgccg gggctccttt 4740 gctctgtgtg ctggggttgg accctgtggg caaaggaggc ttgtgctgtt ttggggggtc 4800 gaggaggctg gaatgtgcgg aagccactgt gtgggtggtg agggcgcgct gggctgaggc 4860 acctggctca tctcttcatg ggcagccgag gtgtggtggc tgggccatgc actactctgt 4920 gacctcctgg gagccatcca gccgctcttc tgtcagcctg gggcaggctt gggccgacag 4980 tggaggagga gtgggaggtg gctcagggct aagtctggga atcccatccc tgcccccacc 5040 accccacatc tgcacattct gccttggctc atacactctt tccagctggg ggacaggtac 5100 aggtaggtct tcccacccac agctgaggag tcagggtttg gcttaagcaa gccagcttca 5160 gtattctata ggacaacagg ctggggggac agacctggcc caaggccata ggtgagctgg 5220 tggctgcaga gctgggactg gcccctgtca ggtgtgcccc atgcacgtgt gagtttgtac 5280 cctggggcag tgttgctcat gctgtgtgca gactcagggg tagggtcact gctgttaggg 5340 gtggtcctga cacctccgcc cagaggcttg acctctcagg cacagagaag agccctgtcc 5400 Pagina 95

P318031NL_ST25{9969840} (1) aggggcagct accttcttct gcccccttat cagcaggggc ctggccagtg ttttettttt 5460 tttttttcag tccccacttg atcactgaag ggccacccac ccatgggcat ttgctctctg 5520 ccttaaggag ctgggcactt agacagcaga cagagcagaa atggagcctg tgagcctgtc 5580 catgtgttcc ctccctgccc taggcctcag ctgctcccgc agcccatctg gtaccgctgg 5640 aggccctgtg tccaccccag ggtggtcaaa tctttgacta tgacccgtct tgacttctag 5700 ttatttcctc taaggagatg ttctcccagc agcgccatct gtgaaggtgt cttgggcaag 5760 cccagcttga gtatgaaggg gagtctttgg gactcatcaa ctatggcctc cccaacccaa 5820 aagcctgagg gtgtgggggc ctgggagtca gtgcttctgg gagcagggcc agcacctctg 5880 gaattcatgg gggctgggca aggccttggg ttcagctggc tgcccaggga tggcagggag 5940 agcttgggga gggggetttt gggttgagct gcatgcctga gagaggattc tagggagacc 6000 cagaagcttc tgggacagtg tggagaagct ggccggagct cttcttagtt tgccttggga 6060 gcaagaggga cgtgcctgct gtcaccgggt ccggatggcc agcctgcctc tgtctgatga 6120 ctgtggagga ggcagagtgt gagtgtgagc tggtacaggg gctgtgtgtg tcactgctga 6180 gcagtccctg tgtgcacgtc cctctgtata agttgggtgc catctcctcc cagctgggct 6240 atgtcttctt tgtccctttg taagagggtg ggaggatgag gactgtgggc tgtcccggac 6300 ctgtgtggag agggggctga gggagcgagg tgtgcgagtg gagggtggga gtgtcttggt 6360 ctttctccat cctggttgcc tggtgtcctc gggcctgtgg catgaagagg ggctggtaag 6420 aactgcagtg actaccagtc agtggattgg ggctatggct cctggggcca ctgcccaggt 6480 tgggagctgt tgtccttgga accctttccg gtctgagact atggcgtgtg tggttgggtg 6540 tgtgggccag cgtctcaggg cagtcacagt ggcaggtctg catctgcatt gcecggeggge 6600 agcagcactg ggtgttcctg ccgagtggcc tgaatccacg tctgccggga aatgttggaa 6660 ctgtttggag ccagtcctgt gctgtgtggc attttgggaa ccatgatgcc caaacctggg 6720 ggtggccatt tgctgtggtc gcttggctga ggggtgggca ttctgatgga agcttttttt 6780 ggcctcctcc tcattctgct tggccccggc agagatgtca tcgctcctgc ccccgaggtc 6840 ccatggcctg cctcacccac acctccgcct tccccgggtg caggtcctgt ccctgggcgc 6900 cgacgtgctg cctgagtaca agctgcaggc accgcgcatc caccgctgga ccatcctgca 6960 Pagina 96

P318031NL_ST25{9969840} (1) ttacagcccc ttcaaggccg tgtgggactg gctcatcctg ctgctggtca tctacacggc 7020 tgtcttcaca ccctactcgg ctgccttcct gctgaaggag acggaagaag gcccgcctgc 7080 taccgagtgt ggctacgcct gccagccgct ggctgtggtg gacctcatcg tggacatcat 7140 gttcattgtg gacatcctca tcaacttccg caccacctac gtcaatgcca acgaggaggt 7200 ggtcagccac cccggccgca tcgccgtcca ctacttcaag ggctggttcc tcatcgacat 7260 ggtggccgcc atcccctteg acctgctcat cttcggctct ggctctgagg aggtggggtc 7320 agcaaggagg caggtggtgt agggagagga gaggccagga atgtggtggg aggtagtggg 7380 gtggagggge tggcgaggag gacagagaaa aagagagaga gagagaaaga gagaaagaca 7440 gtgggagaga gacatggtgg caaggcagct gcagaggcaa ggtgggtacc ccagcaaacc 7500 tatgctcctt ctctccacac aagctcctcg caggttgcag agggccacac aggacacctt 7560 tttttctaat tcgtccatcc caaggggcca ttattttgta gcgtcaccaa agtgccctag 7620 aagctggtgg gagtgggcag gcaccctagg gtgatcactg cacctgtcag tgcccctgtt 7680 tcctcatcca cactatgggg gctgcctctg tcccaaagct agcactaaga ttaatgagac 7740 acctgtgctg ggcccagtgt gggcttcacc tcttaggagg agggtctagg aagtctttgg 7800 ggatctgacc tctgatgctc gctctgaggg ccgcagcacc tcccctcctc ccttgccccc 7860 cttgccccat caacggaatg tgccccttcc ctgtccccca gctgatcggg ctgctgaaga 7920 ctgcgcggct gctgcggctg gtgcgcgtgg cgcggaagct ggatcgctac tcagagtacg 7980 gcgcggccgt gctgttcttg ctcatgtgca cctttgcgct catcgcgcac tggctagcct 8040 gcatctggta cgccatcggc aacatggagc agccacacat ggactcacgc atcggctggc 8100 tgcacaacct gggcgaccag ataggcaaac cctacaacag cagcggcctg ggcggcccct 8160 ccatcaagga caagtatgtg acggcgctct acttcacctt cagcagcctc accagtgtgg 8220 gcttcggcaa cgtctctccc aacaccaact cagagaagat cttctccatc tgcgtcatgc 8280 tcattggctg tgagtgtgcc CaggggCgEgg Cggcggggag agcccacggt ggaggaaccc 8340 aagttggagg aaactgaggc tgctagccgg gccagagtga ggccctggaa cttagagact 8400 ccaggggcca ccatcgtgac atggtttggg ggctgggtca ccagggcggg gactactgaa 8460 gegtggggtg tgctggagcg cagatgtaca agggcgtctc gtgggcatgt cgacgctgag 8520 Pagina 97

P318031NL_ST25{9969840} (1) actgagacac tgacctggtg cggggcctga gcagggtccc tgtgggtggg tggggtccct 8580 gcagaggctg acggccccac gtgcccacgc ccccagccct catgtatgct agcatcttcg 8640 gcaacgtgtc ggccatcatc cagcggctgt actcgggcac agcccgctac cacacacaga 8700 tgctgcgggt gcgggagttc atccgcttcc accagatccc caatcccctg Cgccagcgcc 8760 tcgaggagta cttccagcac gcctggtcct acaccaacgg catcgacatg aacgcggtga 8820 ggccaccaga gcgtggccag tgggtggcag gctgggaaga gtggggtggc agaggggaac 8880 ctcttggcac agcaaacaag tctcaggcgg tcacagtcag tagtaaggac cctggaggca 8940 cgttgcccct ttttgcccat gaggaaatgg aagcccttgg aaccccttcc ctggagagct 9000 ccccttttgg ggtgggaggt atatgtcaca taggcgatgg ggttaaatcc ttgtgacagc 9060 ccagggcggg aatggtctct atgccttaca aataagaaca tggaggtcta gagcccaagg 9120 gtgctcggcc aggcatggtg aagctggagt ggggtcccag gtccttccca agacactggg 9180 ccccctgatg cttccgagat ctcccaggcc tggaggttga gatttctctg acatggaggg 9240 gtgggatggt ggagtagagt gtgggttggg gggtcccaag ggagggtgtg ctgagctgcc 9300 cccaccctgc ccccaggtgc tgaagggctt ccctgagtgc ctgcaggctg acatctgcct 9360 gcacctgaac cgctcactgc tgcagcactg caaacccttc cgaggggcca ccaagggctg 9420 ccttcgggcc ctggccatga agttcaagac cacacatgca ccgccagggg acacactggt 9480 gcatgctggg gacctgctca ccgccctgta cttcatctcc cggggctcca tcgagatcct 9540 gcggggcgac gtcgtcgtgg ccatcctggg tatggggtgg ggggcgggca ctggactgga 9600 aatgccctct gcagcctcaa gaggtgcgag ccttctgaat atgcagtcac tggggctgtg 9660 gacctgggac tgcctgcagg gtcactgggc tcctttaatt cacctaaact caggccctcc 9720 aagcggggcc atggagagga gccccacgtg gggtgaggct gctgaactct ggggttccca 9780 cattctcctt cccttcagga tccgccacaa acagacactt tttgcttcct taaagtagga 9840 tcaaatctag atcctctagc ctgggcagta gaggaagaaa tgctagcctg gaagctcggc 9900 atttggtttc actaagggcc atgtggttcc ctgcagcctc atgcctggcc ccttgacaca 9960 tccaaagcaa agggagtcct gccccctccc cccacttcct ttctaccctg cctgtgcaca 10020 gtgggtgggt tggtgtgtct ggacactgag gacttcctcc ccctttgect gtccttcect 10080 Pagina 98

P318031NL_ST25{9969840} (1) cggccctgtg tgcctcaggg cagatatagc aagctctttc gaccatagtt gatggtagga 10140 cattttagac tttgtttctc agctctgtac aaacacaaat acacaccccc acaaaactaa 10200 aatcaaagtt tcactacata acactgggcc ttactgcatg tggttcattc tagcatttct 10260 gttctgtgct gtgctaagct atactactgt atgttctttc agtaaaaaaa aaaaaaaaaa 10320 aaaaaaaatg ctggttttga ttcactactg tgttctgatc tttggtttga agaacattgc 10380 ttataagggt gcagtgattg gctaagaggg tgtttgggac ctggggtttg gggtagtcct 10440 gctggctggg tctcatttgc tgggagaccc tgggctcctg gcccctctgg cactgtcgtg 10500 cctgctggga gctgagggag ggtgggetgg cagattgctt ccccggttgt gtgggttggg 10560 ggccggctgg caggcgaccc agctgagggg acatgctctg cccggggtgg ggatgeeggg 10620 agaaggtgcc tgctgcctgg atgccgcccc ccggggctga gctccctgtc ctctccatgg 10680 cctccaggga agaatgacat ctttggggag cctctgaacc tgtatgcaag gcctggcaag 10740 tcgaacgggg atgtgcgggc cctcacctac tgtgacctac acaagatcca tcgggacgac 10800 ctgctggagg tgctggacat gtaccctgag ttctccgacc acttctggtc cagcctggag 10860 atcaccttca acctgcgaga tgtgagttgg ctgccctgag ctggacccca tcctcctgct 10920 tccagctgtg tggctgcccc tttgctgtgt gacattgaat gtgatgcttc tctgtctcag 10980 tttccctgtc tgtcaaatgg tatcatagag cagcctacaa aagtcccaaa ctcagtcttt 11040 gcctggggca aaatcacatt gggggcaggg aagggttttc ccagagctgg ggcgcccagc 11100 cctacttttt ctgtgttaag gagggagctt ggggcctgac cccggtgggg caggagagcea 11160 ctgaaagggc cctgatactg attttggttc cagaccaaca tgatcccggg ctcccccggc 11220 agtacggagt tagagggtgg cttcagtcgg caacgcaagc gcaagttgtc cttccgcagg 11280 cgcacggaca agggtgaggc gggggagees aggaagggEg AggECgggga Caaggtgagg 11340 ctgggagctg gaaggcgggg tgggccgtgt ctgctttacc caggcctcag accttcccat 11400 cccttcctcc agtgtgccca ctccacaccc cagctgtcca gatgctgccc atctttcagg 11460 gcccctgcag ctgtcacctc ctccaggaag ccctcttcca ccctccaggg caagcctagg 11520 ctttttggac ttcacagggc tggggtctct gtgagcactg aggaggaaag aacatagaac 11580 aaaggagggc cagggagcca acgatggggc tgaggccccg cagtcccaga gatccgccaa 11640 Pagina 99

P318031NL_ST25{9969840} (1) cccctgaagc accattgcca gtgcttgggc agtcaggggg tcctgggagt tcttggtcgg 11700 cttgacagtg ggtgcaagat agcagaagaa gcgactgggg acaaaccagg gcttcctgcc 11760 cagtcctctc tcgggctcgc tcctgtcccc tcctccaccc tcgccccctec ctetetgttce 11820 tcctcccctc tctgaggccc attctctgtt tcccacagac acggagcagc caggggaggt 11880 gtcggccttg gggeegggec gSgCEgSEgC agggccgagt agCCggggcc ggeegggsss 11940 gccgtggggg gagagcccgt ccagtggccc ctccagccct gagagcagtg aggatgaggg 12000 cccaggccgc agctccagcc ccctccgcct ggtgcccttc tccagcccca ggCCCCCCgg 12060 agagccgccg ggtggggagc ccctgatgga ggactgcgag aagagcagcg acacttgcaa 12120 ccccctgtca ggtatccCCgg gCgaCggEgCg ggcgagggag gaccgggcgt ggcagcggtg 12180 gtgcgtctac cccgctcacc cagctctgct ctctggctgc aggcgccttc tcaggagtgt 12240 ccaacatttt cagcttctgg ggggacagtc ggggccgcca gtaccaggag ctccctcgat 12300 gccccgcccc cacccccagc ctcctcaaca tccccctctc cagcccgggt Cggcggcccc 12360 ggggcgacgt ggagagcagg ctggatgccc tccagcgcca gctcaacagg tgagggagtg 12420 caggtggggt gggggggcac gccctggagt ctggtccagg tcctggcggt gttgtctggt 12480 agagggagag ggcctgtctg tgtgtgcagc agctggatcc ccttcttcca ttcctagccc 12540 agctgggggc tgccacacct ctagcgcgga gggcctgggt tgacaggctg cccctccccc 12600 ctccccttcc ccttccccac tcccggtgga ggctgtcact ggtgtcccca cttctctgag 12660 catcccccac ttcctgcccc aggctggaga cccggctgag tgcagacatg gccactgtcc 12720 tgcagctgct acagaggcag atgacgctgg tcccgcccgc ctacagtgct gtgaccaccc 12780 cggggcctgg ccccacttcc acatccccgc tgttgcccgt cagccccctc cccaccctca 12840 ccttggactc gctttctcag gtaagctcca gccctgcttc ctcgatgtac cgtgacccgc 12900 ttgttccccg attccagccc aaacctgctt tgctgccttt ctgctgggct ctgtggccca 12960 gcctccctga accttgttcc tgccctcctc ccacccctgt cctcctggag gaaggctgct 13020 tcaggagctg caggctcagg gtagggcagg agagtccccc gtccaaagcc cctagacagc 13080 tgggtgcagg gaacctgtag gctaagtggc ccgcagcttc actcccctag tgtccacagc 13140 ctccaccgcc aggccccgga gcatcgcctg aagagccagc tccaacggtg gtgcctgtct 13200 Pagina 109

P318031NL_ST25{9969840} (1) cacggccacg ggtggtctcc aagaactgac tgagcccgag acgtggcggt ggaggctgca 13260 taccagagta tctagggaca gggtcccggg tcccttctgt gagtgacact ccctggtctc 13320 accgtcctgc ccatccttgc ccttggcagt ggctgtcaca gggtcatctt gagggtgctt 13380 ggcatttccc agggtgcaga ggccaagcct ccaggggagg acttcctggg cctgctttgc 13440 cccctctgct agagatgggc cttagttgcc ccttgtcctc tcctgggtgg acctgcatgt 13500 tcctggcctc tgcaggatgg cggtgtctgt gtcaacctcc gcgtccatgg tggggactcc 13560 tctggctgcc agaggcggcc tgggatctgc tccctcccca cttcccttgg catccgctcc 13620 cccctcccat atgctgcctg ctcctgtttg cagacacgag ggcccacgtc agcaaatgtg 13680 ccagtctccc ttcgcagccg cccccgcaga gctctcacca gcagacgctc agtacccaca 13740 aacccctccc gcctgccccg gccctctccc tggccttcag atgcctggcc gccagcctgc 13800 ctctgtttct aggtccccga gttctttctg gattgcggga aggaatcttg tctcttttte 13860 tgtcccagct tctgtcccaa cacagagact gcttgttcca ggttgggttg ggctcagcgg 13920 ccacattccc tcgggaagag acaggggcct agggttttcc ctgctcccag ctctcttgtt 13980 tggctcctgc tctcagagaa tgcctttctcec tacttcccac cttggtgcct ccccttggtg 14040 gcccctgtcc tgctctegct gccttggctc ttgtcccecctc cagcttctct gcccacctga 14100 ctctcctctt ggccctcctg tcctccegtc catcctctgt ccccgggttc tcctgcccect 14160 ttecctcccc ttcctcctcc tccatggcct cttcgcctgc ccatgctctg tgtgtattgc 14220 aggtttccca gttcatggcg tgtgaggagc tgcccccggg ggccccagag cttccccaag 14280 aaggccccac acgacgcctc tccctaccgg gccagctggg ggccctcacc tcccagcccc 14340 tgcacagaca cggctcggac ccgggcagtt agtggggctg cccagtgtgg acacgtggct 14400 cacccaggga tcaaggcgct gctgggccgc tccccttgga ggccctgctc aggaggccct 14460 gaccgtggaa ggggagagga actcgaaagc acagctcctc ccccagccct tgggaccatc 14520 ttctcctgca gtcccctggg ccccagtgag aggggcaggg gcagggccgg caglaggtgg 14580 ggcctgtggt ccccccactg ccctgagggc attagctggt ctaactgccc ggaggcaccc 14640 ggccctgggc cttaggcacc tcaaggactt ttctgctatt tactgctctt attgttaagg 14700 ataataatta aggatcatat gaataattaa tgaagatgct gatgactatg aataataaat 14760 Pagina 101

P318031NL_ST25{9969840} (1) aattatcctg aggagaactc ca 14782 <210> 191 <211> 2880 <212> DNA <213> Artificial Sequence <220> <223> Sequence of Bxbl attB fragment introduced into Bac RP11-10L20 by recombineering <400> 191 cttaattccc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 60 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc 120 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc 180 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct 240 ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag 300 gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc 360 acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg 420 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg 480 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc 540 gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat 600 ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag 660 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct 720 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac 780 cttegcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct 840 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact 900 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt 960 tcggecgagge ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct 1020 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggeggea 1080 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct 1140 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca 1200 Pagina 102

P318031NL_ST25{9969840} (1) caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg 1260 gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg 1320 geggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg 1380 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg 14409 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga 1500 ggaattcgcc accatggccg gcttgtcgac gacggcggtc tccgtcgtca ggatcatccg 1560 gatcccacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 1620 ggtgaggaac gccaccatgg gatcggccat tgaacaagat ggattgcacg caggttctcc 1680 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 1740 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 1800 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 1860 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 1920 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1980 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 2040 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 2100 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 2160 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 2220 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 2280 gegtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 2340 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 2400 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 2460 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 2520 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 2580 ggggatctca tgctggagtt cttcgcccac cccccggatc taagctctag ataagtaatg 2640 atcataatca gccatatcac atctgtagag gttttacttg ctttaaaaaa cctcccacac 2700 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 2760 Pagina 103

P318031NL_ST25{9969840} (1) gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 2820 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 2880 <210> 192 <211> 8284 <212> DNA <213> Artificial Sequence <220> <223> pBR-attB ASAP2f plasmid <400> 192 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta 240 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 300 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 360 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 420 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc 480 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct 540 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 600 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggegeg 660 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 720 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 780 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc 840 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc 900 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg 960 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc 1020 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg 1080 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta 1140

Pagina 104

P318031NL_ST25{9969840} (1) ttteggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttegg 1200 CgaggCgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc 1260 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gCggcaaggc 1320 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag 1380 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa 14409 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag Laccgggcgc 1500 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggges 1560 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag 1620 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca 1680 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa 1740 ttegccacca tggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccggatc 1800 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg 1860 acggggtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 1920 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 1980 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 2040 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 2100 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 2160 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 2220 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 2280 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 2340 tgcagcgatg ggSSCggESE g8eeeeecgc gegeraggrg SEECEESECE gegegcrgagsses 2400 CEEEECELEL Ccgaggcggag aggtgcecggeg gcagccaatc agagcggcgc gctccgaaag 2460 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag CgCgCgECgg 2520 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct CCgCCgCCgC CtCgCgccgc 2580 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct 2640 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt 2700 Pagina 105

P318031NL_ST25{9969840} (1) gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc gSggggtgcg 2760 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag 2820 CgCLgCgggc gcggcgcggg gctttgtgcg ctccgcagtg LgCgCgaggg gagegeggec 2880 BEgggcggtg ccccgeggtyg cgggggegggce tgcgagggga acaaaggctg Cgtgcggggt 2940 gtgtgcgtgg gggggtgage agggggtgtg ggcgcgtcgg tcgggctgca accccccctg 3000 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg 3060 cgtggcgcgg ggctcgccgt gCCgSgCggg gggtggegge aggtgggggt BCCBSECEES 3120 gcggggeege CtCgggCCgg ggagggetcg ggggageggee gCggCggcC CCggagcgcc 3180 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg 3240 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gCgccgccgc 3300 accccctcta gegggegegg ggcgaagegg tgeggegecg gcaggaagga aatgggeges 3360 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt 3420 ccgegggggg acggctgcct TCggggggga Cggggcaggg cggggttcgg cttctggcgt 3480 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag 3540 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgag 3600 gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc 3660 ctggctcaca aataccactg agatctgcat gagctagccg ccaccatgga gacgactgtg 3720 aggtatgaac aggggtcaga gctcactaaa acttcgagct ctccaacagc agatgagccc 3780 acgataaaga ttgatgatgg tcgtgatgag ggtaatgaac aagacagctg ttccaatacc 3840 attaggagaa aaatttcccc gtttgtgatg tcatttggat tcagagtatt tggagttgtg 3900 cttatcattg tagacatcat agtggtgatt gtggatctgg ccatcagtga gaagaaaaga 3960 ggcattagag agattcttga aggtgtttcc ctggctatag cactcttctt ccttgttgat 4020 gttctcatga gagtgtttgt tgaaggcttc aagaactatt tccggtccaa actgaatact 4080 ttggatgcag tcatagtagt gggcactctg ctaattaata tgacctactc cttctctgac 4140 cttagcttta acagccataa cgtgtatatt accgcggata aacagaaaaa cggcattaaa 4200 gcgaacttta ccgtgcgcca taacgtggaa gatggcagcg tgcagctggc ggatcattat 4260 Pagina 106

P318031NL_ST25{9969840} (1) cagcagaaca ccccgattgg cgatggcccg gtgctgctgc cggataacca ttatctgagc 4320 acccagaccg tgctgagcaa agatccgaac gaaaaacgcg atcacatggt gctgctggaa 4380 tttgtgaccg cagcgggcat tacacacggc atggatgaac tgtatggcgg caccggceggc 4440 agcgcgagcc agggcgaaga actgtttacc ggcgtggtgc cgattctggt ggaactggat 4500 ggcgatgtga acggccataa atttagcgtg cgcggcgaag gcgaaggcga tgcgaccatt 4560 ggcaaactga ccctgaaatt tatttgcacc accggcaaac taccggtgcc gtggccgacc 4620 ctggtgacca ccttaaccta tggcgtgcag tgctttagcc gctatccgga tcatatgaaa 4680 cgccatgatt tttttaaaag cgcgatgccg gaaggctatg tgcaggaacg caccattagc 4740 tttaaagatg atggcaaata taaaacccgc gcggtggtga aatttgaagg cgataccctg 4800 gtgaaccgca ttgaactgaa aggcaccgat tttaaagaag atggcaacat tctggggcat 4860 aaactggaat ataacacaga tcagatgccg cagatggtta ctcttcttcg agttctgaga 4920 attgttatct taataagaat atttcgcctg gcttcacaga agaaacaact tgaagtggta 4980 acctaatgaa agcttggtac cgagctcgga tccactagtc cagtgtggtg gaattctgca 5040 gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag 5100 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgecttect 5160 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 5220 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggges 5280 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 5340 cggaactagc gctgcaagaa ctcttcctca cgataacttc gtataggaga ctttatacga 5400 agttaagcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 5460 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 5520 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 5580 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 5640 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 5700 gctacacttg ccagcgccct agcgcccgct cctttegctt tcttcectte ctttetegec 5760 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 5820 Pagina 107

P318031NL_ST25{9969840} (1) agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 5880 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 5940 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 6000 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 6060 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 6120 tgctctgatg ccgcatagtt aagccagccc cgacacccge caacacccgc tgacgcgccc 6180 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 6240 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 6300 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 6360 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 6420 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 6480 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 6540 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 6600 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 6660 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 6720 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 6780 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 6840 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 6900 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 6960 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 7020 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 7080 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 7140 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 7200 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 7260 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 7320 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 7380 Pagina 108

P318031NL_ST25{9969840} (1) gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 7440 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 7500 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 7560 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 7620 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 7680 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 7740 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 7800 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 7860 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 7920 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 7980 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 8040 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 8100 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 8160 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 8220 gctgataccg CtCgCCgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaageg 8280 gaag 8284 <210> 193 <211> 9460 <212> DNA <213> Artificial Sequence <220> <223> pBR-attB jRCaMPlb plasmid <400> 193 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata 60 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta 120 atgtactggg gttgaacact ctgtgccatt ggctccggtg cccgtcagtg ggcagagcgc 180 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag 240 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc 300

Pagina 109

P318031NL_ST25{9969840} (1) gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac 360 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt 420 acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt 480 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc 540 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc 600 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt 660 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat 720 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag 780 cgcacatgtt CggCgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag 840 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc 900 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc 960 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggegggtgag 1020 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg 1080 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct 1140 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact 1200 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt 1260 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag 1320 gtgtcgtgag gaattcgcca ccatggccgg cttgtcgacg acggcggtct ccgtcgtcag 1380 gatcatccgg atccataact tcgtatagca tacattatac gaagttatca tgatattcgg 14409 caagcaggca tcgtagctgc cgatgtatca cctctcgcaa gaattcaagc ttggaggccg 1500 cgaattctga aagaccccac ctgtaggttt ggcaagccca gggatgtacg tccctaaccc 1560 gctagggggc agcaactagt cccaggcctg cactgccgcc tgccggcagg ggtccagtcg 1620 ctagcgcatg cctgcactac tggagttccg cgttacataa cttacggtaa atggcccgcc 1680 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 1740 aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt aaactgccca 1800 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 1860 Pagina 110

P318031NL_ST25{9969840} (1) taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 1920 gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc acgttctgct 1980 tcactctccc catctcccce ccctccccac ccccaatttt gtatttattt attttttaat 2040 tattttgtgc agcgatgggg SCgSESEESE SESEESCECE cgccaggrgg SECSEZECSE 2100 ggcgaggggc SEBSCESEEC gaggeggaga ggtgeggegg cagccaatca gagcggcgcg 2160 ctccgaaagt ttccttttat ggCgaggcgg cggcggcggc ggccctataa aaagcgaagc 2220 gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg 2280 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagegg gcgggacggc 2340 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg 2400 ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt geggggggga geggeteggs 2460 gegtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct gCCCggCggC 2520 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gegtgtgege gaggggageg 2580 cggeeggggg cggtgccccg cggtgegggg gggctgcgag gggaacaaag gctgcgtgcg 2640 gggtgtgtge gtgggggget gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc 2700 ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg ggectccgtg 2760 cggggegtgg cgcggggctc gCCgtgCCgg gCgSSEgSEtg geggraggtg ggggtgecgg 2820 gcggggeggg gCCgCCTCgg geeggggagg getcggggga BSEECECBEC ggCCCCggag 2880 cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga 2940 gagggcgcag ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc 3000 cgcaccccct ctagegggeg cgggegaage ggtgeggege Cggcaggaag gaaatgggcg 3060 gegagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag cctcggggct 3120 gCCgCagggg gacggctgcc tTCggggggg acggggcagg gcggggttcg gettetggeg 3180 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcaaaca 3240 gctcctgggc aacgtgctgg ttattgtact gtctcatcat tttggcaaag aattccgatc 3300 gcgccaccat gctgcagaac gagcttgctc ttaagttggc tggacttgat attaacaaga 3360 ctggaggagg ttctcatcat catcatcatc atggtatggc tagcatgact ggtggacagc 3420 Pagina 111

P318031NL_ST25{9969840} (1) aaatgggtcg ggatctgtac gacgatgacg ataaggatct cgcaacaatg gtcgactcat 3480 cgcgacgtaa gtggaataag tggggtcacg cagtcagagc tataggtcgg ctgagctcag 3540 cgaacaacac cgaaatgatg tacccagcgg atggtggtct gcgtggttac actcacatgg 3600 cgctgaaagt tgatggcggc ggtcacctgt cctgttcttt cgtgaccacc taccgctcca 3660 aaaagactgt cggcaacatt aagatgcctg ccattcatta cgtcagccac cgtctggagc 3720 gcctggagga gagcgataac gaaatgtttg tcgtacagcg tgaacacgca gttgccaagt 3780 ttgtgggcct gggtggtggc ggcggtaccg gagggagcat gaactccctg atcaaggaga 3840 acatgcgtat gaaagtggtt ctggaaggct ccgtaaacgg ccaccagttc aaatgcactg 3900 gtgaaggcga aggcaacccg tatatgggca cccagactat gcgtatcaaa gtgatcgagg 3960 gtggtccgct gccgtttgcg ttcgacatcc tggcgacgtc ctttatgtat ggctcccgta 4020 ccttcatcaa atatccgaaa ggcatcccgg atttctttaa gcagtccttc ccggaaggtt 4080 ttacctggga acgtgtgacc cgttacgagg acggcggcgt aattaccgtt atgcaagaca 4140 cgtctctgga ggatggctgc ctggtgtatc acgtgcaggt tcgcggtgtg aacttcccga 4200 gcaatggtgc tgtaatgcaa aagaaaacca aaggttggga gcctacggac tcccaactga 4260 ctgaagagca gatcgcagaa tttaaagagg ctttctccct atttgacaag gacggggatg 4320 ggacaataac aaccaaggag atggggacgg tgatgcggtc cctggggcag aaccccacag 4380 aagcagagct gcaggacatg atcaatgaag tagatgccga cggtgacggc acaatcgact 4440 tccctgagtt cctgattatg atggcaggca aaatgaaata cacagacagt gaagaagaaa 4500 ttagagaagc gttcggcgtg tttgataagg atggcaatgg ctacatcagt gcagcagagc 4560 ttcgccacgt gatgacaaac cttggagaga agttaacaga tgaagaggtt gatgaaatga 4620 tcagggaagc agacagcgat ggggatggtc aggtaaacta cgaagagttt gtacaaatga 4680 tgacagcgaa gtaggtaaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc 4740 tggtgtggcc aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa 4800 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat 4860 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag 4920 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca 4980 Pagina 112

P318031NL_ST25{9969840} (1) tatgctggct gccatgaaca aaggtggcta taaagaggtc atcagtatat gaaacagccc 5040 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata 5100 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta 5160 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatga 5220 agatcggatc gctactagag gatgcacatg tgaccgaggg aggagctgca agaactcttc 5280 ctcacgataa cttcgtatag gagactttat acgaagttaa gcgctcactg gccgtcgttt 5340 tacagggaca cgaagtgatc cgtttaaact atcagtgttt gacaggatat attggcgggt 5400 aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg cgtgaaaagg 5460 tttatccgtt cgtccatttg tatgtgccag ccgcctttgc gacgctcacc gggctggttg 5520 ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa cgcgccagaa acgccgtcga 5580 agccgtgtgc gagacaccgc ggccgccggc gttgtggata cctcgcggaa aacttggccc 5640 tcactgacag atgaggggcg gacgttgaca cttgaggggc cgactcaccc ggcgcggcgt 5700 tgacagatga ggggcaggct cgatttcggc cggcgacgtg gagctggcca gcctcgcaaa 5760 tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat gatgtggaca agcctgggga 5820 taagtgccct gcggtattga cacttgaggg gcgcgactac tgacagatga ggggcgcgat 5880 ccttgacact tgaggggcag agtgctgaca gatgaggggc gcacctattg acatttgagg 5940 ggctgtccac aggcagaaaa tccagcattt gcaagggttt ccgcccgttt ttcggccacc 6000 gctaacctgt cttttaacct gcttttaaac caatatttat aaaccttgtt tttaaccagg 6060 gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg tgccccccct tctcgaaccc 6120 tcccggcccg ctaacgcggg cctcccatcc ccccaggggc tgcgcccctc ggccgcgaac 6180 ggcctcaccc caaaaatggc agcgctggcc aattcccgag tgcgcggaac ccctatttgt 6240 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 6300 cttcaataat attgaaaaag gaagagtatg gctaaaatga gaatatcacc ggaattgaaa 6360 aaactgatcg aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta 6420 tataagctgg tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa 6480 gggaccacct atgatgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg 6540 Pagina 113

P318031NL_ST25{9969840} (1) cctgttccaa aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt 6600 gaggccgatg gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt 6660 atcgagctgt atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc 6720 tatacgaata gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg 6780 gccgatgtgg attgcgaaaa ctgggaagag gacactccat ttaaagatcc gcgcgagctg 6840 tatgattttt taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg 6900 ggagacagca acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga 6960 agcggcaggg cggacaagtg gtatgacatt gccttctgcg tccggtcgct cagggaggat 7020 atcggggaag aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg 7080 gagaaaataa aatattatat tttactggat gaattgtttt agctgtcaga ccaagtttac 7140 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 7200 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 7260 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 7320 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 7380 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 7440 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 7500 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 7560 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 7620 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 7680 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 7740 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 7800 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 7860 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggcagat 7920 cctagatgtg gcgcaacgat gccggcgaca agcaggagcg caccgacttc ttccgcatca 7980 agtgttttgg ctctcaggcc gaggcccacg gcaagtattt gggcaagggg tcgctggtat 8040 tcgtgcaggg caagattcgg aataccaagt acgagaagga cggccagacg gtctacggga 8100 Pagina 114

P318031NL_ST25{9969840} (1)

ccgacttcat tgccgataag gtggattatc tggacaccaa ggcaccaggc gggtcaaatc 8160 aggaataagg gcacattgcc ccggcgtgag tcggggcaat cccgcaagga gggtgaatga 8220 atcggacgtt tgaccggaag gcatacaggc aagaactgat cgacgcgggg ttttccgccg 8280 aggatgccga aaccatcgca agccgcaccg tcatgcgtgc gccccgcgaa accttccagt 8340 ccgtcggctc gatggtccag caagctacgg ccaagatcga gcgcgacagc gtgcaactgg 8400 ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg ttcgcgtcgt cttgaacagg 8460 aggcggcagg tttggcgaag tcgatgacca tcgacacgcg aggaactatg acgaccaaga 8520 agcgaaaaac CgCCggCgag gacctggcaa aacaggtcag cgaggccaag caggccgcgt 8580 tgctgaaaca cacgaagcag cagatcaagg aaatgcagct ttccttgttc gatattgcgc 8640 cgtggccgga cacgatgcga gcgatgccaa acgacacggc ccgctctgcc ctgttcacca 8700 cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa ggtcattttc cacgtcaaca 8760 aggacgtgaa gatcacctac accggcgtcg agctgcgggc cgacgatgac gaactggtgt 8820 ggcagcaggt gttggagtac gcgaagcgca cccctatcgg cgagccgatc accttcacgt 8880 tctacgagct ttgccaggac ctgggctggt cgatcaatgg ccggtattac acgaaggccg 8940 aggaatgcct gtcgcgccta caggcgacgg cgatgggctt cacgtccgac cgcgttgggc 9000 acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct ggaccgtggc aagaaaacgt 9060 cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct gtttgctggc gaccactaca 9120 cgaaattcat atgggagaag taccgcaagc tgtcgccgac ggcccgacgg atgttcgact 9180 atttcagctc gcaccgggag ccgtacccgc tcaagctgga aaccttccgc ctcatgtgcg 9240 gatcggattc cacccgcgtg aagaagtggc gcgagcaggt cggcgaagcc tgcgaagagt 9300 tgcgaggcag cggcctggtg gaacacgcct gggtcaatga tgacctggtg cattgcaaac 9360 gctagggcct tgtggggtca gttccggctg ggggttcagc agcccctgct cggatctgtt 9420 ggaccggaca gtagtcatgg ttgatgggct gcctgtatcg 9460 <210> 194

<211> 8699

<212> DNA

<213> Artificial Sequence

Pagina 115

P318031NL_ST25{9969840} (1) <220> <223> pBR-attB Lck-miRFP703 plasmid <400> 194 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata 60 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta 120 atgtactggg gttgaacact ctgtgccatt ggctccggtg cccgtcagtg ggcagagcgc 180 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag 240 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc 300 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac 360 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt 420 acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt 480 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc 540 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc 600 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt 660 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat 720 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag 780 cgcacatgtt CggCgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag 840 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc 900 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc 960 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggegggtgag 1020 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg 1080 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct 1140 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact 1200 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt 1260 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag 1320 gtgtcgtgag gaattcgcca ccatggccgg cttgtcgacg acggcggtct ccgtcgtcag 1380 gatcatccgg atccataact tcgtatagca tacattatac gaagttatca tgatattcgg 14409

Pagina 116

P318031NL_ST25{9969840} (1) caagcaggca tcgtagctgc cgatgtatca cctctcgcaa ggttttgacg ggtttactag 1500 aattcgagct cggagactaa ctacggtgaa agagacttag aaggagattg agagatactg 1560 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 1620 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 1680 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 1740 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 1800 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 1860 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc 1920 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 1980 8888888888 BEECECECEC CAgBCEEEEC BEBECEEEEC BIgEEECEEE EBCEEEECEAE 2040 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc 2100 gaggCggCgg cggcggcggc cctataaaaa gcgaagcgcg CggCgggCgg gagtcgctgc 2160 gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc ggctctgact 2220 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 2280 gcgcttggtt taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct 2340 ccgggaggge cctttgtgcg ggggggageg gctcgggggg tgcgtgcgtg tgtgtgtgcg 2400 tggggagcgc cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg BgCgCggCgC 2460 ggggctttgt gcgctccgcg tgtgecgegag gggagegegeg CCggggECgg TgCCCCgCgEg 2520 TgCggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg SggSggtgag 2580 cagggggtgt gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt 2640 gctgagcacg gcccggcttc gggtgegggg ctccgtacgg ggegtggege ggggctcgcc 2700 gtgccgggeg ggggetggeg gCaggTgEgg grgecgggeg gggeggggec gcctcgggcc 2760 ggggagggct cgggggagegg grgeggrgge ccccggageg ccggcggctg tCgaggcgcg 2820 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg 2880 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagCcgggcgc 2940 ggggcgaage ggtgeggege Cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg 3000 Pagina 117

P318031NL_ST25{9969840} (1) ccgcgccgcc gtccccttct ccctctccag cctcggggct gtececgegggg ggacggctgc 3060 cttcggggegg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga 3120 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg 3180 gttattgtgc tgtctcatca ttttggcaaa gaattccgat cgcgccacca tgggctgcgt 3240 gtgcagcagc aaccccgagc taccggtcgc cacaatggta gcaggtcatg cctctggcag 3300 ccccgcattc gggaccgcct ctcattcgaa ttgcgaacat gaagagatcc acctcgccgg 3360 ctcgatccag ccgcatggcg cgcttctggt cgtcagcgaa catgatcatc gcgtcatcca 3420 ggccagcgcc aacgccgcgg aatttctgaa tctcggaagc gtactcggcg ttccgctcgc 3480 cgagatcgac ggcgatctgt tgatcaagat cctgccgcat ctcgatccca ccgccgaagg 3540 catgccggtc gcggtgcgct gccggatcgg caatccctct acggagtact gcggtctgat 3600 gcatcggcct ccggaaggcg ggctgatcat cgaactcgaa cgtgccggcc cgtcgatcga 3660 tctgtcaggc acgctggcgc cggcgctgga gcggatccgc acggcgggtt cactgcgcgc 3720 gctgtgcgat gacaccgtgc tgctgtttca gcagtgcacc ggctacgacc gggtgatggt 3780 gtatcgtttc gatgagcaag gccacggcct ggtattctcc gagtgccatg tgcctgggct 3840 cgaatcctat ttcggcaacc gctatccgtc gtcgctggtc ccgcagatgg cgcggcagct 3900 gtacgtgcgg cagcgcgtcc gcgtgctggt cgacgtcacc tatcagccgg tgccgctgga 3960 gccgcggctg tcgccgctga ccgggcgcga tctcgacatg tcgggctgct tcctgcgctc 4020 gatgtcgccg atccatctgc agttcctgaa ggacatgggc gtgcgcgcca ccctggcggt 4080 gtcgctggtg gtcggcggca agctgtgggg cctggttgtc tgtcaccatt atctgccgcg 4140 cttcatccgt ttcgagctgc gggcgatctg caaacggctc gccgaaagga tcgcgacgcg 4200 gatcaccgcg cttgagagct aagcggccgc gactctagat cataatcagc cataccacat 4260 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata 4320 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa 4380 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 4440 tgtccaaact catcaatgta tcttaaggcg ctttacgagg atgcacatgt gaccgaggga 4500 ggagctgcaa gaactcttcc tcacgataac ttcgtatagg agactttata cgaagttaag 4560 Pagina 118

P318031NL_ST25{9969840} (1) cgctcactgg ccgtcgtttt acagggacac gaagtgatcc gtttaaacta tcagtgtttg 4620 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aatcggatat 4680 ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgccagc cgcctttgcg 4740 acgctcaccg ggctggttgc cctcgccgct gggctggcgg ccgtctatgg ccctgcaaac 4800 gcgccagaaa cgccgtcgaa gccgtgtgcg agacaccgcg gccgccggcg ttgtggatac 4860 ctcgcggaaa acttggccct cactgacaga tgaggggcgg acgttgacac ttgaggggcc 4920 gactcacccg gcgcggcgtt gacagatgag gggcaggctc gatttcggcc ggcgacgtgg 4980 agctggccag cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt cccacagatg 5040 atgtggacaa gcctggggat aagtgccctg cggtattgac acttgagggg cgcgactact 5100 gacagatgag gggcgcgatc cttgacactt gaggggcaga gtgctgacag atgaggggcg 5160 cacctattga catttgaggg gctgtccaca ggcagaaaat ccagcatttg caagggtttc 5220 cgcccgtttt tcggccaccg ctaacctgtc ttttaacctg cttttaaacc aatatttata 5280 aaccttgttt ttaaccaggg ctgcgccctg tgcgcgtgac cgcgcacgcc gaaggggggt 5340 gccccccctt ctcgaaccct cccggcccgc taacgcgggc ctcccatccc cccaggggct 5400 gcgcccctcg gccgcgaacg gcctcacccc aaaaatggca gcgctggcca attcccgagt 5460 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 5520 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatgg ctaaaatgag 5580 aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag atacggaagg 5640 aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat atttaaaaat 5700 gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg acatgatgct 5760 atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc atgatggctg 5820 gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt atgaagatga 5880 acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct ttcactccat 5940 cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg aattggatta 6000 cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagagg acactccatt 6060 taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag aggaacttgt 6120 Pagina 119

P318031NL_ST25{9969840} (1) cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca aagtaagtgg 6180 ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg ccttctgcgt 6240 ccggtcgctc agggaggata tcggggaaga acagtatgtc gagctatttt ttgacttact 6300 ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg aattgtttta 6360 gctgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 6420 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 6480 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6540 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6600 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6660 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6720 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6780 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6840 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6900 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6960 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 7020 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 7080 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 7140 tttacggttc ctggcagatc ctagatgtgg cgcaacgatg CCggCgacaa gCaggagcgc 7200 accgacttct tccgcatcaa gtgttttggc tctcaggccg aggcccacgg caagtatttg 7260 ggcaaggggt cgctggtatt cgtgcagggc aagattcgga ataccaagta cgagaaggac 7320 ggccagacgg tctacgggac cgacttcatt gccgataagg tggattatct ggacaccaag 7380 gcaccaggcg ggtcaaatca ggaataaggg cacattgccc cggcgtgagt cggggcaatc 7440 ccgcaaggag ggtgaatgaa tcggacgttt gaccggaagg catacaggca agaactgatc 7500 gacgcggggt tttccgccga ggatgccgaa accatcgcaa gccgcaccgt catgcgtgcg 7560 ccccgcgaaa ccttccagtc cgtcggctcg atggtccagc aagctacggc caagatcgag 7620 cgcgacagcg tgcaactggc tccccctgcc ctgcccgcgc catcggccgc cgtggagcgt 7680 Pagina 120

P318031NL_ST25{9969840} (1) tcgcgtcgtc ttgaacagga ggcggcaggt ttggcgaagt cgatgaccat cgacacgcga 7740 ggaactatga cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa acaggtcagc 7800 gaggccaagc aggccgcgtt gctgaaacac acgaagcagc agatcaagga aatgcagctt 7860 tccttgttcg atattgcgcc gtggccggac acgatgcgag cgatgccaaa cgacacggcc 7920 cgctctgccc tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct gcaaaacaag 7980 gtcattttcc acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga gctgcgggcc 8040 gacgatgacg aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac ccctatcggc 8100 gagccgatca ccttcacgtt ctacgagctt tgccaggacc tgggctggtc gatcaatggc 8160 cggtattaca cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc gatgggcttc 8220 acgtccgacc gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt ccgcgtcctg 8280 gaccgtggca agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat cgtcgtgctg 8340 tttgctggcg accactacac gaaattcata tgggagaagt accgcaagct gtcgccgacg 8400 gcccgacgga tgttcgacta tttcagctcg caccgggagc cgtacccgct caagctggaa 8460 accttccgcc tcatgtgcgg atcggattcc acccgcgtga agaagtggcg cgagcaggtc 8520 ggcgaagcct gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg ggtcaatgat 8580 gacctggtgc attgcaaacg ctagggcctt gtggggtcag ttccggctgg gggttcagca 8640 gcccctgctc ggatctgttg gaccggacag tagtcatggt tgatgggctg cctgtatcg 8699 <210> 195 <211> 19526 <212> DNA <213> Artificial Sequence <220> <223> pBR-attB AJMA donor plasmid <400> 195 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata 60 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta 120 atgtactggg gttgaacact ctgtgccatt ggctccggtg cccgtcagtg ggcagagcgc 180 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag 240

Pagina 121

P318031NL_ST25{9969840} (1) agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc 300 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac 360 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt 420 acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt 480 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc 540 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc 600 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt 660 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat 720 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag 780 cgcacatgtt CggCgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag 840 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc 900 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc 960 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggegggtgag 1020 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg 1080 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct 1140 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact 1200 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt 1260 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag 1320 gtgtcgtgag gaattcgcca ccatggccgg cttgtcgacg acggcggtct ccgtcgtcag 1380 gatcatccgg atccataact tcgtatagca tacattatac gaagttatca tgatattcgg 14409 caagcaggca tcgtagctgc cgaattcgga tccggagact aactacggtg aaagagactt 1500 agaaggagat tgagagatac tggagttccg cgttacataa cttacggtaa atggcccgcc 1560 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 1620 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 1680 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 1740 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 1800 Pagina 122

P318031NL_ST25{9969840} (1) gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt 1860 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 1920 attttgtgca gcgatggggg CSggSESEES GEESECSCEC gecaggrggg BCEESECEES 1980 BCgaggggcg SEgCEESECg aggeggagag gLgCggcggc agccaatcag agcggcgcgc 2040 tccgaaagtt tccttttatg gCgaggcggc ggcggcggcg gccctataaa aagcgaagcg 2100 cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc 2160 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgageggg Cgggacggcc 2220 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc 2280 tgcgtgaaag ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggeteggge 2340 ggtgegtgeg tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct 2400 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cgtgtgegeg aggggagcgc 2460 ggccggggge ggtgccccgc gSLgCggggg SECTgCgagg ggaacaaagg ctgegtgegg 2520 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc 2580 cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac 2640 ggggcgtggc gcggggctcg CCggCCggg CggSgESTgg CggCaggtgg gggtgccggg 2700 cggggcrgggg ccgecteggg ccggggaggg ctcgggggag gggcgeggeg gCCCCCggag 2760 cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga 2820 gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg 2880 ccgcaccccc tctageggge gCggggCgaa geggtgegge gCCggcagga aggaaatggg 2940 cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg 3000 ctgtccgegg ggggacgget gCctLCgggEg gggacgggge agggcggggt tcggcttctg 3060 gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttect 3120 acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattccg 3180 atcgcgccac catggagacg actgtgaggt atgaacaggg gtcagagctc actaaaactt 3240 cgagctctcc aacagcagat gagcccacga taaagattga tgatggtcgt gatgagggta 3300 atgaacaaga cagctgttcc aataccatta ggagaaaaat ttccccgttt gtgatgtcat 3360 Pagina 123

P318031NL_ST25{9969840} (1) ttggattcag agtatttgga gttgtgctta tcattgtaga catcatagtg gtgattgtgg 3420 atctggccat cagtgagaag aaaagaggca ttagagagat tcttgaaggt gtttccctgg 3480 ctatagcact cttcttcctt gttgatgttc tcatgagagt gtttgttgaa ggcttcaaga 3540 actatttccg gtccaaactg aatactttgg atgcagtcat agtagtgggc actctgctaa 3600 ttaatatgac ctactccttc tctgacctta gctttaacag ccataacgtg tatattaccg 3660 cggataaaca gaaaaacggc attaaagcga actttaccgt gcgccataac gtggaagatg 3720 gcagcgtgca gctggcggat cattatcagc agaacacccc gattggcgat ggcccggtgc 3780 tgctgccgga taaccattat ctgagcaccc agaccgtgct gagcaaagat ccgaacgaaa 3840 aacgcgatca catggtgctg ctggaatttg tgaccgcagc gggcattaca cacggcatgg 3900 atgaactgta tggcggcacc ggcggcageg cgagccaggg cgaagaactg tttaccggcg 3960 tggtgccgat tctggtggaa ctggatggcg atgtgaacgg ccataaattt agcgtgcgcg 4020 gcgaaggcga aggcgatgcg accattggca aactgaccct gaaatttatt tgcaccaccg 4080 gcaaactacc ggtgccgtgg ccgaccctgg tgaccacctt aacctatggc gtgcagtgct 4140 ttagccgcta tccggatcat atgaaacgcc atgatttttt taaaagcgcg atgccggaag 4200 gctatgtgca ggaacgcacc attagcttta aagatgatgg caaatataaa acCCCgCgCgg 4260 tggtgaaatt tgaaggcgat accctggtga accgcattga actgaaaggc accgatttta 4320 aagaagatgg caacattctg gggcataaac tggaatataa cacagatcag atgccgcaga 4380 tggttactct tcttcgagtt ctgagaattg ttatcttaat aagaatattt cgcctggctt 4440 cacagaagaa acaacttgaa gtggtaacct aggtaaattc actcctcagg tgcaggctgc 4500 ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct 4560 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg 4620 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact 4680 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag 4740 tttggcaaca tatgccatat gctggctgcc atgaacaaag gtggctataa agaggtcatc 4800 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag 4860 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt 4920 Pagina 124

P318031NL_ST25{9969840} (1) ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg 4980 tccctcttct cttatgaaga tcggatcgct gcaagaattc aagcttggag actaactacg 5040 gtgaaagaga cttagaagga gattgagaga tactggagtt ccgcgttaca taacttacgg 5100 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 5160 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 5220 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 5280 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 5340 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc 5400 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat 5460 ttatttttta attattttgt gcagcgatgg gEECEggEEEE BEEBEZEZECE CECEgCCaggC 5520 £888CgeeLeC ggeecgages gCgeeecges gcgaggegga gaggtgegge ggcagccaat 5580 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat 5640 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgttgc cttcgccccg tgccccgctc 5700 cgcgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc 5760 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggctcgtt 5820 tcttttctgt ggctgcgtga aagccttaaa gggctccggg agggcccttt gLgCggggEgEg 5880 gagcggctcg gggggtgcgt gcgtgtgtgt gtgegtgggg agcgccgcgt gcggcccgcg 5940 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc 6000 gcgaggggag CgCggCCggg ggcggtgccc cgeggtgegg gggggrtgeg aggggaacaa 6060 aggctgcgtg cggggtgtgt gegtggggeg gtgagcaggg ggtgtgggeg cggeggtegg 6120 gctgtaaccc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg 6180 cggggctccg tacggggcgt ggegegggge tcgccgtgcc gggegggggg tggeggragy 6240 tgggggtgec ggSCggggcg gggccgcctc gggCCgggga SEECTCgEEE gageggecscsy 6300 gCggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat 6360 ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat 6420 CLgggaggcg ccgccgcacc ccctctagcg ZgCgCgggEC gaagcggtgc ggegeceggea 6480 Pagina 125

P318031NL_ST25{9969840} (1) ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc 6540 tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagegsces 6600 ggttcggett ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct 6660 tettettttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg 6720 gcaaagaatt ccgatcgcgc caccatgctg cagaacgagc ttgctcttaa gttggctgga 6780 cttgatatta acaagactgg aggaggttct catcatcatc atcatcatgg tatggctagc 6840 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatctcgca 6900 acaatggtcg actcatcgcg acgtaagtgg aataagtggg gtcacgcagt cagagctata 6960 ggtcggctga gctcagcgaa caacaccgaa atgatgtacc cagcggatgg tggtctgcgt 7020 ggttacactc acatggcgct gaaagttgat ggcggcggtc acctgtcctg ttctttcgtg 7080 accacctacc gctccaaaaa gactgtcggc aacattaaga tgcctgccat tcattacgtc 7140 agccaccgtc tggagcgcct ggaggagagc gataacgaaa tgtttgtcgt acagcgtgaa 7200 cacgcagttg ccaagtttgt gggcctgggt ggtggcggcg gtaccggagg gagcatgaac 7260 tccctgatca aggagaacat gcgtatgaaa gtggttctgg aaggctccgt aaacggccac 7320 cagttcaaat gcactggtga aggcgaaggc aacccgtata tgggcaccca gactatgcgt 7380 atcaaagtga tcgagggtgg tccgctgccg tttgcgttcg acatcctggc gacgtccttt 7440 atgtatggct cccgtacctt catcaaatat ccgaaaggca tcccggattt ctttaagcag 7500 tccttcccgg aaggttttac ctgggaacgt gtgacccgtt acgaggacgg cggcgtaatt 7560 accgttatgc aagacacgtc tctggaggat ggctgcctgg tgtatcacgt gcaggttcgc 7620 ggtgtgaact tcccgagcaa tggtgctgta atgcaaaaga aaaccaaagg ttgggagcct 7680 acggactccc aactgactga agagcagatc gcagaattta aagaggcttt ctccctattt 7740 gacaaggacg gggatgggac aataacaacc aaggagatgg ggacggtgat gcggtccctg 7800 gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt 7860 gacggcacaa tcgacttccc tgagttcctg attatgatgg caggcaaaat gaaatacaca 7920 gacagtgaag aagaaattag agaagcgttc ggcgtgtttg ataaggatgg caatggctac 7980 atcagtgcag cagagcttcg ccacgtgatg acaaaccttg gagagaagtt aacagatgaa 8040 Pagina 126

P318031NL_ST25{9969840} (1) gaggttgatg aaatgatcag ggaagcagac agcgatgggg atggtcaggt aaactacgaa 8100 gagtttgtac aaatgatgac agcgaagtag gtaaattcac tcctcaggtg caggctgcct 8160 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt 8220 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct 8280 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg 8340 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt 8400 tggcaacata tgccatatgc tggctgccat gaacaaaggt ggctataaag aggtcatcag 8460 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt 8520 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc 8580 ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc 8640 cctcttctct tatgaagatc ggatcgctac tagaattcga gctcggagac taactacggt 8700 gaaagagact tagaaggaga ttgagagata ctggagttcc gcgttacata acttacggta 8760 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 8820 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 8880 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 8940 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 9000 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc 9060 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 9120 attttttaat tattttgtgc agcgatgggg EBCEgggEZEEE GEgEBEECECE CECCaAgECEE 9180 BECgESECSE SECSAgSEEC GEESCESESC gaggeggaga gSTgCggCgg cagccaatca 9240 gagcggcgcg ctccgaaagt ttccttttat ggCgaggcgg cggcggcggc ggccctataa 9300 aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg 9360 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg 9420 gcgggacgge ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc 9480 ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt geggggggga 9540 gcggctcggg gggtgegtge gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct 9600 Pagina 127

P318031NL_ST25{9969840} (1) gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc 9660 Baggggagcg CggCCggggg Cggtgccccg cggtgegggg gggctgegag gggaacaaag 9720 gctgcgtgcg gggtgtgtgc gLgggggegt Sagcaggggg tgtgggegeg geggteggsc 9780 tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 9840 gggctccgta cggggcgtgg cgcggggctc gecgtgecgg gCggSgggtg gCggcaggtg 9900 ggggtgecgg geggggrgeg geegectegg gecggggagg getcggggga GEESCECgEC 9960 ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 10020 taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 10080 gggaggcgee gccgcacccc ctctageggg CgCggggcga ageggtgegg CgCCggCagg 10140 aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctcte 10200 cagcctcggg gctgtccgcg gggggacgge tgccttcCggg ggggacgggg CaggSCggEg 10260 tteggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 10320 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc 10380 aaagaattcc gatcgcgcca ccatgggctg cgtgtgcagc agcaaccccg agctaccggt 10440 cgccacaatg gtagcaggtc atgcctctgg cagccccgca ttcgggaccg cctctcattc 10500 gaattgcgaa catgaagaga tccacctcgc cggctcgatc cagccgcatg gcgcgcttct 10560 ggtcgtcagc gaacatgatc atcgcgtcat ccaggccagc gccaacgccg cggaatttct 10620 gaatctcgga agcgtactcg gcgttccgct cgccgagatc gacggcgatc tgttgatcaa 10680 gatcctgccg catctcgatc ccaccgccga aggcatgccg gtcgcggtgc gctgccggat 10740 cggcaatccc tctacggagt actgcggtct gatgcatcgg cctccggaag gcgggctgat 10800 catcgaactc gaacgtgccg gcccgtcgat cgatctgtca ggcacgctgg cgccggcgct 10860 ggagcggatc cgcacggcgg gttcactgcg cgcgctgtgc gatgacaccg tgctgctgtt 10920 tcagcagtgc accggctacg accgggtgat ggtgtatcgt ttcgatgagc aaggccacgg 10980 cctggtattc tccgagtgcc atgtgcctgg gctcgaatcc tatttcggca accgctatcc 11040 gtcgtcgctg gtcccgcaga tggcgcggca gctgtacgtg cggcagcgcg tccgcgtgct 11100 ggtcgacgtc acctatcagc cggtgccgct ggagccgcgg ctgtcgccgc tgaccgggeg 11160 Pagina 128

P318031NL_ST25{9969840} (1) cgatctcgac atgtcgggct gcttcctgcg ctcgatgtcg ccgatccatc tgcagttcct 11220 gaaggacatg ggcgtgcgcg ccaccctggc ggtgtcgctg gtggtcggcg gcaagctgtg 11280 gegcctggtt gtctgtcacc attatctgcc gcgcttcatc cgtttcgagc tgcgggcgat 11340 ctgcaaacgg ctcgccgaaa ggatcgcgac gcggatcacc gcgcttgaga gctaagcggc 11400 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa 11460 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 11520 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 11580 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag 11640 gcgctttacg aattcccatg gggaggccgc gaattctgaa agaccccacc tgtaggtttg 11700 gcaagcccag ggatgtacgt ccctaacccg ctagggggca gcaactagtc ccaggcctgc 11760 actgccgcct gccggcaggg gtccagtcgc tagcgcatgc ctgcactact ggagttccgc 11820 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 11880 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 11940 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 12000 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 12060 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 12120 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc 12180 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gEgEEEZEEZEE 12240 88BBCECECE CCagECEEEE CBEEECEEEE CEABEBEECEE BECEEEECEA BECEEABAEE 12300 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cCgaggcggcg 12360 gcggeggegg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc 12420 gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 12480 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt 12540 ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg 12600 ccctttgtge gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gLggggagcg 12660 ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggetttg 12720 Pagina 129

P318031NL_ST25{9969840} (1) tgcgctccgc gtgtgcgcga ggggagegeg gccgggggeg gtgccccgcg grgeggesss 12780 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gCagggggtg 12840 tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac 12900 ggcccggctt cgggtgcggg gctccgtacg gggegtggeg cggggctcgc cgtgeegggc 12960 g8ggggtggc ggrcaggtggg gETSCCHESC ggggrgggee CgCCTCgZEC CEEZZaAggEC 13020 TCEgggEgagE BECECgECEEg CCCCCggagC gccggcggct gtecgaggege ggegagecgc 13080 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc 13140 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagegggeg cggggegaag 13200 CggLgCggCg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc 13260 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg CCLTCgggEEg 13320 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct 13380 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg 13440 ctgtctcatc attttggcaa agaattccga tcgcgccacc atggagacga ctgtgaggta 13500 tgaacagggg tcagagctca ctaaaacttc gagctctcca acagcagatg agcccacgat 13560 aaagattgat gatggtcgtg atgagggtaa tgaacaagac agctgttcca ataccattag 13620 gagaaaaatt tccccgtttg tgatgtcatt tggattcaga gtatttggag ttgtgcttat 13680 cattgtagac atcatagtgg tgattgtgga tctggccatc agtgagaaga aaagaggcat 13740 tagagagatt cttgaaggtg tttccctggc tatagcactc ttcttccttg ttgatgttct 13800 catgagagtg tttgttgaag gcttcaagaa ctatttccgg tccaaactga atactttgga 13860 tgcagtcata gtagtgggca ctctgctaat taatatgacc tactccttct ctgaccttag 13920 ctttaacagc cataacgtgt atattaccgc ggataaacag aaaaacggca ttaaagcgaa 13980 ctttaccgtg cgccataacg tggaagatgg cagcgtgcag ctggcggatc attatcagca 14040 gaacaccccg attggcgatg gcccggtgct gctgccggat aaccattatc tgagcaccca 14100 gaccgtgctg agcaaagatc cgaacgaaaa acgcgatcac atggtgctgc tggaatttgt 14160 gaccgcagcg ggcattacac acggcatgga tgaactgtat ggcggcaccg gCggcagcgc 14220 gagccagggc gaagaactgt ttaccggcgt ggtgccgatt ctggtggaac tggatggcga 14280 Pagina 130

P318031NL_ST25{9969840} (1) tgtgaacggc cataaattta gcgtgecgegg cgaaggcgaa ggcgatgcga ccattggcaa 14340 actgaccctg aaatttattt gcaccaccgg caaactaccg gtgccgtggc cgaccctggt 14400 gaccacctta acctatggcg tgcagtgctt tagccgctat ccggatcata tgaaacgcca 14460 tgattttttt aaaagcgcga tgccggaagg ctatgtgcag gaacgcacca ttagctttaa 14520 agatgatggc aaatataaaa cccgcgcggt ggtgaaattt gaaggcgata ccctggtgaa 14580 ccgcattgaa ctgaaaggca ccgattttaa agaagatggc aacattctgg ggcataaact 14640 ggaatataac acagatcaga tgccgcagat ggttactctt cttcgagttc tgagaattgt 14700 tatcttaata agaatatttc gcctggcttc acagaagaaa caacttgaag tggtaaccta 14760 ggtaaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat 14820 gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc 14880 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata 14940 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta 15000 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgccatatg ctggctgcca 15060 tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc 15120 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt 15180 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct 15240 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat cggatcgctc 15300 agagaggatg cacatgtgac cgagggagga gctgcaagaa ctcttcctca cgataacttc 15360 gtataggaga ctttatacga agttaagcgc tcactggccg tcgttttaca gggacacgaa 15420 gtgatccgtt taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa 15480 agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc 15540 catttgtatg tgccagccgc ctttgcgacg ctcaccgggc tggttgccct cgccgctggg 15600 ctggcggccg tctatggccc tgcaaacgcg ccagaaacgc cgtcgaagcc gtgtgcgaga 15660 caccgcggcc gccggcgttg tggatacctc gcggaaaact tggccctcac tgacagatga 15720 geggcggacg ttgacacttg aggggccgac tcacccggcg cggcgttgac agatgagggg 15780 caggctcgat ttcggccggc gacgtggagc tggccagcct cgcaaatcgg cgaaaacgcc 15840 Pagina 131

P318031NL_ST25{9969840} (1) tgattttacg cgagtttccc acagatgatg tggacaagcc tggggataag tgccctgcgg 15900 tattgacact tgaggggcgc gactactgac agatgagggg cgcgatcctt gacacttgag 15960 gggcagagtg ctgacagatg aggggcgcac ctattgacat ttgaggggct gtccacaggc 16020 agaaaatcca gcatttgcaa gggtttccgc ccgtttttcg gccaccgcta acctgtcttt 16080 taacctgctt ttaaaccaat atttataaac cttgttttta accagggctg cgccctgtgc 16140 gcgtgaccgc gcacgccgaa ggggggtgcc cccccttctc gaaccctccc ggcccgctaa 16200 cgcgggcctc ccatcccccc aggggctgcg cccctcggcc gcgaacggcc tcaccccaaa 16260 aatggcagcg ctggccaatt cccgagtgcg cggaacccct atttgtttat ttttctaaat 16320 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 16380 aaaaaggaag agtatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa 16440 ataccgctgc gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg 16500 agaaaatgaa aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga 16560 tgtggaacgg gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt 16620 cctgcacttt gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt 16680 cctttgctcg gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc 16740 ggagtgcatc aggctctttc actccatcga catatcggat tgtccctata cgaatagctt 16800 agacagccgc ttagccgaat tggattactt actgaataac gatctggccg atgtggattg 16860 cgaaaactgg gaagaggaca ctccatttaa agatccgcgc gagctgtatg attttttaaa 16920 gacggaaaag cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat 16980 ctttgtgaaa gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga 17040 caagtggtat gacattgcct tctgcgtccg gtcgctcagg gaggatatcg gggaagaaca 17100 gtatgtcgag ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata 17160 ttatatttta ctggatgaat tgttttagct gtcagaccaa gtttactcat atatacttta 17220 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 17280 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 17340 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 17400 Pagina 132

P318031NL_ST25{9969840} (1) aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 17460 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 17520 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 17580 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 17640 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 17700 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 17760 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 17820 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 17880 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 17940 atggaaaaac gccagcaacg cggccttttt acggttcctg gcagatccta gatgtggcgc 18000 aacgatgccg gcgacaagca ggagcgcacc gacttcttcc gcatcaagtg ttttggctct 18060 caggccgagg cccacggcaa gtatttgggc aaggggtcgc tggtattcgt gcagggcaag 18120 attcggaata ccaagtacga gaaggacggc cagacggtct acgggaccga cttcattgcc 18180 gataaggtgg attatctgga caccaaggca ccaggcgggt caaatcagga ataagggcac 18240 attgccccgg cgtgagtcgg ggcaatcccg caaggagggt gaatgaatcg gacgtttgac 18300 cggaaggcat acaggcaaga actgatcgac gcggggtttt ccgccgagga tgccgaaacc 18360 atcgcaagcc gcaccgtcat gcgtgcgccc cgcgaaacct tccagtccgt cggctcgatg 18420 gtccagcaag ctacggccaa gatcgagcgc gacagcgtgc aactggctcc ccctgccctg 18480 cccgcgccat cggccgccgt ggagcgttcg cgtcgtcttg aacaggaggc ggcaggtttg 18540 gcgaagtcga tgaccatcga cacgcgagga actatgacga ccaagaagcg aaaaaccgcc 18600 ggcgaggacc tggcaaaaca ggtcagcgag gccaagcagg ccgcgttgct gaaacacacg 18660 aagcagcaga tcaaggaaat gcagctttcc ttgttcgata ttgcgccgtg gccggacacg 18720 atgcgagcga tgccaaacga cacggcccgc tctgccctgt tcaccacgcg caacaagaaa 18780 atcccgcgcg aggcgctgca aaacaaggtc attttccacg tcaacaagga cgtgaagatc 18840 acctacaccg gcgtcgagct gcgggccgac gatgacgaac tggtgtggca gcaggtgttg 18900 gagtacgcga agcgcacccc tatcggcgag ccgatcacct tcacgttcta cgagctttgc 18960 Pagina 133

P318031NL_ST25{9969840} (1) caggacctgg gctggtcgat caatggccgg tattacacga aggccgagga atgcctgtcg 19020 cgcctacagg cgacggcgat gggcttcacg tccgaccgcg ttgggcacct ggaatcggtg 19080 tcgctgctgc accgcttccg cgtcctggac cgtggcaaga aaacgtcccg ttgccaggtc 19140 ctgatcgacg aggaaatcgt cgtgctgttt gctggcgacc actacacgaa attcatatgg 19200 gagaagtacc gcaagctgtc gccgacggcc cgacggatgt tcgactattt cagctcgcac 19260 cgggagccgt acccgctcaa gctggaaacc ttccgcctca tgtgcggatc ggattccacc 19320 cgcgtgaaga agtggcgcga gcaggtcggc gaagcctgcg aagagttgeg aggcagcggc 19380 ctggtggaac acgcctgggt caatgatgac ctggtgcatt gcaaacgcta gggccttgtg 19440 gggtcagttc cggctggggg ttcagcagcc cctgctcgga tctgttggac cggacagtag 19500 tcatggttga tgggctgcct gtatcg 19526 <210> 196 <211> 5064 <212> DNA <213> Artificial Sequence <220> <223> KCNH2-Bxb1-LP-TC plasmid <400> 196 ccggttcaga caggataaag aggaacgcag aatgttagac aacacccgct tacgcatagc 60 tattcagaaa tcaggccgtt taagcgatga ttcacgagaa ttgctggccc gctgcggcat 120 aaaaattaat ttacacactc agcgctgatg aatcccctaa tgattttggt aaaaatcatt 180 aagttaaggt ggacacacat cttgtcatat gattaaatgg tttcgcgaaa aatcaataat 240 cagacaacaa gatgtgcgaa ctcgatattt tacacgactc tctttaccaa ttctgccccg 300 aattacactt aaaacgactc aacagcttaa cgttggcttg ccacgcatta cttgactgta 360 aaactctcac tcttaccgaa cttggccgta acctgccaac caaagcgaga acaaaacata 420 acatcaaacg aatcgaccga ttgttaggta atcgtcacct gcaggaaggt ttaaacgcat 480 ttaggtgaca ctatagaagt gtgtatcgct cgagggatcc gaattcagga ggtaaaaacc 540 atgataggtg ccctgtcttg ggctctgaag gccctacata caaagtcatc ctatctgctg 600 aggcaaagat ataaggttac ctcgtgtaaa acgacggcca gtcgctagcg agctcggcgc 660

Pagina 134

P318031NL_ST25{9969840} (1) gccataactt cgtatagcat acattatacg aagttagaat tcgccttctg caggagcgta 720 cagaacccag ggccctggca cccgtgctac cgggtagggg aggcgctttt cccaaggcag 780 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg 840 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc 900 ttegcgccac cttccactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg 960 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc 1020 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc 1080 ttegctttct gggctcagag gctgggaagg ggtgggtccg BgggCggEct CaggggCgEgE 1140 ctcaggggecg gggegggege ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa 1200 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagg 1260 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 1320 tggacggcga cgtaaacggc cacaagttca gcCgtgtcCgg Cgagggcgag ggcgatgcca 1380 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 14409 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 1500 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 1560 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 1620 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 1680 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 1740 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 1800 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 1860 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 1920 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 1980 agtaactgca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 2040 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 2100 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctaga 2160 tatacgtggt ttgtctggtc aaccaccgcg gtctcagtgg tgtacggtac aaacccatat 2220 Pagina 135

P318031NL_ST25{9969840} (1) gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac 2280 agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc 2340 atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg 2400 ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat 2460 gagaacaggg gcatcttgag cccctgcgga cggtgtcgac aggtgcttct cgatctgcat 2520 cctgggatca aagcgatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt 2580 gaattgctgc cctctggtta tgtgtgggag ggctaaatta ccctgttatc cctacctcga 2640 ctgtgccttc tacagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 2700 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 2760 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggLggggc 2820 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 2880 ctatggcttc tgaggcataa cttcgtatag gagactttat acgaagttaa tggatcccct 2940 atagagcgta cccagcacag tcagcacaac cagatggcca tgtgctccgg ggccagaggs 3000 agacggaggg acactgtggc cctgataata attaattaag acgtcagaat tctcgaggcg 3060 gccgcatgtg cgtctcccta tagtgagtcg tattaatttc gcgggcggaa cccctatttg 3120 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 3180 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 3240 teectttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 3300 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 3360 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 3420 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 3480 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 3540 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 3600 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 3660 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 3720 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 3780 Pagina 136

P318031NL_ST25{9969840} (1) attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 3840 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 3900 taaatctgga gccggtgagc gtggatctcg cggtatcatt gcagcactgg ggccagatgg 3960 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 4020 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 4080 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 4140 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 4200 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 4260 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 4320 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 4380 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 4440 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 4500 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 4560 geggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 4620 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 4680 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 4740 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 4800 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaat gcggcctttt tacggttcct 4860 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 4920 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagceg 4980 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 5040 gcgttggccg attcattaat gcag 5064 <210> 197 <211> 54298 <212> DNA <213> Artificial Sequence <220>

Pagina 137

P318031NL_ST25{9969840} (1)

<223> pl5-attB KCNH2 wt donor plasmid

<400> 197 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg 60 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag 120 ttggggggag gegtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg 180 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata 240 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta 300 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct 360 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt 420 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag 480 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc 540 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt 600 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt 660 ggggeegegg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc 720 ctgcgagege ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg 780 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg 840 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa 900 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc 960 ttteegtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac 1020 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat 1080 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg 1140 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct 1200 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tcgccaccat 1260 ggccggcttg tcgacgacgg cggtctccgt cgtcaggatc atccggatcc ataacttcgt 1320 atagcataca ttatacgaag ttatcatgat attcggcaag caggcatcga cttgagacgg 1380 atggttgtca gggatccatc tgacaggccc tgcccccttc ctgcctcctec ctgtctccect 14409 ctcctgccac cctcagcctg gcccagtttc ctggcatggt tgctgtggcg cctcttggga 1500

Pagina 138

P318031NL_ST25{9969840} (1) atcggagctt tgggcctcaa gtgtgcccag catggaaatt ccacagcaac tgtcactggg 1560 atcgtctgga cagaaccctc tcaggtgccc ccaggagggg ctgggccctc tctccagacc 1620 agggtgggag gagcagcctt tagagccccc tgctgatctg tgagatgtgg aaatcctgac 1680 ttcaagccct ggagaacctt cagtcattgg tgacttgtct tttttcctgc tctccccaaa 1740 tggattacat gcccgactcc cagtgacctc ccagaggcgt ccctgacata tttgctgttg 1800 atgatgacct gaggcttccg aggggctccc aggaaatggg gttcccagtc tgtcgtctgg 1860 tttcctaagc accagccccg tctgcgctgg gcactgctga gccgggcact gctgagccgg 1920 gcactgctga gccgggcact gctgagccca cttcctagag gagatgttgt tctgctgcgc 1980 ggatccagaa gggggagctc tcagggcctg ttcttcttca gccgggaaag atcgaatgcg 2040 ctcttcctgg acatttagcg ccccagcatt gcaataggcc tctgccgtca ccttcacacc 2100 ctcactggct ctgtctggcc ctgcactgga ctgctggtcc cccagaggga cagagatgag 2160 gtcctgacca caattctcag ctcagccaaa ttctccctaa gccttcctcc cttgcgaagc 2220 cttcctccct tgtgaagcct tcccttatcc ttctggcctt aaaacgattc cttcagaccc 2280 tgaagtcaaa gcattcatgg tctgtccggg cgccgtggct cacacctgta atcccagcac 2340 cttgggaggc ccaggtggga ggatcgcgtg agcccaggag tttgagacca gcttgggcaa 2400 catggggaga tcctgtctct attttacaaa aatgaataac atttttagaa aaagaaaaag 2460 cattcatggt ctgcaccact tcttagactt gggatctagg actgtgtaac ttctcttgtc 2520 agctgccatt atttgtattt ggctcctgta atattgtcta atcatgggtg aaaatattgt 2580 cttctcaacg aattgcaaat tttgtaaggc cagggactgt attgtatatc atatatttcc 2640 ttaggaacac ttctcagtgg ggggcacata gaaagctctg gataattgtt tgtgggtata 2700 tcagggagaa gatggagtgg gagacctgtt tgtttattgt ttttgttttt gttttttgag 2760 acaaggtctc actctgttac ccaggctgga gtgcagtggt gctatcacgg ctcactgcac 2820 ctgggcatgg tggctcatgc cggtaatccc agtactttag gaggctgagg agggtggatc 2880 acttgagccc aggaggtcaa aaccagactg ggcaacatag tgagaccctg tctctgaaaa 2940 aaaatgcaaa aattagctag gcatgatggc gcacacctgt agtcccagct gcttgggagg 3000 ctgaagtggg aggatcactt gagcccggga ggtcaaggct gcagtgggcc atgattgtgt 3060 Pagina 139

P318031NL_ST25{9969840} (1) cactgcaccc cagcctggga gacagggtga gaccccatct cagaaaaaga aaagttaaca 3120 ggtagctgca gccttgactt acctggctca agcgatcctc ttgcttcatc ttgctaaata 3180 gctgggacta caggtgtgtg ccatcacacc caactaattt tttctttttg tagagatgag 3240 gtctcgctat gttgcctagg ctggtcttga acacctgggc tcaagccatc ctcccgcctc 3300 agcctcccaa agtgctggga ttacaggtgt aagccaccgt tcctgaccat gcaagaccta 3360 tttgaacctg agttctcttc tcaggtcctt ggtgttggct gactcagcag ctgatgatcc 3420 ttgggatggg ctaggatatg aagctagtgt tgggttagca tttttattcc aagctggggt 3480 aggctcttaa ttctgcccga tatcagcaga atgaacagca aaagttctgg agagctgaca 3540 gagcaacctc tgtatctgga gttctctggt ctctctctct ctctcacaca catatctcaa 3600 atttctttag ctcttgttat atgtggtatg taatctccca cctggcctgg tgtggtggaa 3660 aaatcactta actaagaacc aggagacact attttatgtc cagctccatt tctaacttgt 3720 ggcacaacct tgaggcggag tccttgtcac tgtgaatatc tgttttattg tctaatatat 3780 gaaagtgttt gcttctggct ctaatatttc ataaagcaac agaaactccc aaaggaaatc 3840 ccaagagttg caagtgtaaa gttcaggggt ttattcatat cttttcctga tggctgtcag 3900 aggagtgagg gaggaaggca cactctgaaa gcacatgaga catggttttc tcagcattga 3960 ctggcaaacc cagctcttgc ttcaccccgt gctgcctcca ggagctactg tccagctcca 4020 gaggcatttt caggatttgg ggtgaccatc taatgcactg tgtcctgaag agtgcctcag 4080 tcttgtcacc tgggctgccc tccctggttt tctgctgagc ctgccgtaaa gtattttcag 4140 caccttggac agagacaaag ctccaacctg gctggaggca ttgagtagcc aagtgagctt 4200 gggatctcct ccagggccag ctttcctggc aaggttgccc aaagcatgtc ttggttcttc 4260 atatttttta atcctttgaa ggcactcaaa aataaacccc ctaatatatg tcaaaacagt 4320 gcactagttc tgcaaactct cagggcattt tttaaaacaa aaccaaccta ttttctagaa 4380 tagtgttagg tacacattag gtgcttagta aatacattta aaaaggaata catgcaaatt 4440 caaccttcag acaaatgtcc cagcctcctt ataataggat tctttctccc catcagtccc 4500 tcctatatag taattctgga gcttctagta cttgctaagt tttgcagcct cacttgaagc 4560 cccttcttgt gtctaaaaat cccagtcttt aaatcaaagg aacagatgac tattaagttt 4620 Pagina 140

P318031NL_ST25{9969840} (1) tcaggcagag gaaactgaaa ctggcccctt ctctttctca tctgggggta agtatagatt 4680 ggtggggaaa cccacacctg actcctcttg ggatcttgct ttatgaagat gttcaccacc 4740 ttcctttgta cctgagaaag cccttaccat gcagaaggac caaaatcgcc caggaaattt 4800 tccgagctga cccagcccac tcagagccct ccttcttctg aacacccctc cacgtatcct 4860 caacatcacc agattgttct tctgacattt catatccaag ttttttcatg cctattggtt 4920 cttttcccgg ttagataatg aattccctgt gtttgtggtt tccagttaga tgctactgtg 4980 atgcaaacac agcagataaa atgggagatt gatcagtctt gggggcttct atcttgtcct 5040 aaggggtctc ctgcaccact tatttttagt ctgacataaa tatgcaggat ggatcatggt 5100 gctatttgtg gagtaaccat tggctccaag tgcacccagg atgagagagc agaggcaaga 5160 getggaggtg aagatggcta ttgaggagaa acagaggaac cactcatggg tgggtgtgtt 5220 gctgtgtgga attaaggaat cactcactgc tagagctgga cgggatctca gagatcagct 5280 tgtttggtct cctcatttta caggtaagga aagtgagatt tagtggtact cctaaagtta 5340 cacaacaaat gggccctttt ccatcacctc tgacctggtt caagtgttgg gcatctctcc 5400 cttggactat tgtattgctc tatgtggcag atgatatttt cccaaggtgg tcgtgccaat 5460 acatatgctt ttcttccagt gtgacagcaa cacccctcca ctgagaggtg ggggtctgtg 5520 tttgttctcc ttgcatctga tgggtcacga ttacagcaga agttgtttta tgtgattttt 5580 taaggctaag ttattcaagg agatacagct gctgactggt cttctcttgg gacactggcc 5640 cttggaacac agccaccatg ctgtgaggga gccaagtggc cacacagaag ggcgttgtgt 5700 agctgtctgg ccccagtcct agctgaggtc ccagccaaca gccagcacca accaccaaac 5760 gtaagtagat gaaccttcca gtctctagtt tggagtaaag cagagaaaag tggtcctggt 5820 ggaaactacc ccagttgcaa atccatgagc aaaatatatc caactaaacc actggatctt 5880 gggatggctg ttatgcagca ataggtaatg gaaaaacctc atatccacct cattttctcc 5940 aaactattat acacgtttca ttcaatcaac aaacctttct tggataccta ctctttgcca 6000 gggtctgtac aaagacagaa tatttacatt catggattct gcattcagct gccactactg 6060 tttecttact gtttcctgcc actactgtgt cccccttgca gagataataa aattcatagt 6120 cctcatccct ctgcaaatgg tgccagcctg ctgctccact gccaccacat caaattattt 6180 Pagina 141

P318031NL_ST25{9969840} (1) tgtcctcaga cccaatctcc attctcecttt ctttatatat ccttatgttg tctcttctat 6240 ctgcaatgct tctgccctgc cccacctcca acgttcaagg acctgcactg aaatctcctt 6300 ccctggcaag gccctccaag actcctccgg ttgaaattaa ccaacacctc ctcgtaatcc 6360 cacagcactg actgcagact gcagtttctg tctgcccacc gtctccctca aaactcacta 6420 gactgtgaat ccctggagga gggatcctgg cttatccctc ttgtccccca gcagtgtctg 6480 gcacagggcc cagtacttag aaagccccca gtaaacactt gtcgatgcaa ctagtagcag 6540 aaccagaatt agaagtcaga tctcccgaat cctagtctcc agggccttac ttgcttcttt 6600 tgacaccaca aaggcaggga aaatgtccca ccaaaggtgg ggatcaggtt gtccagagaa 6660 gaccttggaa tggaccaggg gttggcagga ttgctacatg tggagctgca gagggcgatg 6720 agtgaagagt gtcctggctc ttggctaggg gtttcaggcg ggttccttcc atcctttctt 6780 ccttetttct ttecttccta cttcccettect ttctatcaat aagccctgat catccacttc 6840 tggaggggaa acatgctgtt tctttgattc ttttcttctc caactccagt tcacatctga 6900 aaaaaatctc tattctgggg acccagttgc acagaaaaac aaaaaaaacc caccgtgttc 6960 aagcatcaag ggacctgctc actctgttct tccttcccca gggtttccca ggaaccattg 7020 ctctgtaacc tgtctctcct gtgcccagcc gactggaact gcttcccaat cccctgtggg 7080 taaccccagg ctctcctggg cctgactttg gtggtgtctt gcctggcccg ctccttgggg 7140 gacctctggc agagtgggct tccatttctc tttctccttc caggatgtcc tggctccatg 7200 ccttcggact gagcaggagc agagagcagg gtgggtagga tgctttctgc agacttttct 7260 ccctaaattg tttctttttg ggagctggga ggagcctgga tatctcactg tagcctctct 7320 gttccctcag tcacaggcag ggtgcgggag agctcttccc aggaatggtc tctgaaatgg 7380 ctcttggagc tgagatgggg gtggggcaga ttctagaggc ttaacagccc agctcttggt 7440 attagggttg ggcacttttc tctttgtccc agatctgggg gtagcagagg tgggaagttg 7500 aaggcagtgg gggcttctcc atcagctcat ctgaggccca gaaatgttgc tttggggaga 7560 gcttgccccc tctcaccgtt tctcttcctc agctcaattc ttgagtgtgg gggcggctaa 7620 gtgggtctgc cttcaggctc taccctgagc ctccaaggaa gtcagaaatc tacattcagc 7680 tttgacttct gagtatgtgg agtgggctgg ggggagagag ggagagatct gtctctcctc 7740 Pagina 142

P318031NL_ST25{9969840} (1) agcttgaggg aacagctctc ttcttgcctc tgtgcacaga aggagggcta ccccgtgcct 7800 gggctcctct ctgtttggga ggatgaaggg ctcctctgac tcctgtcttc cctttgggat 7860 aagagcagag gcctgtccca gcaacacggg tacaggagtg gaggacagct ggccagctca 7920 gatgagtggc tccttccctt cagggcctgg ctctccctct cccttggtct ggagagaggg 7980 ggaggctaca gatgtccctt tcccttcecttg ccctcccatg cccattctca gctgctggcc 8040 tttgctcact ttctctccat cccctggcct ggacaggagg acacttcact cacacatgaa 8100 tcatcctgga ttcatggtag gcaacccagc agccaggggt ggecttgggg CgLggCggEEg 8160 gctgggccag tcgtggacta ggttctaccc ggaacccaga tgtgttgatg cacctgatgc 8220 caaatgctac accaaggagg agcttgtgct gtggaaggcc ctgggccccc gggggggtga 8280 gtgaggaaac tgctggcagt ggagagggcc tgaggaataa ctgcacaggc aagagcctct 8340 ggactttttt agcatcagct gtggagccac ctccttetct cccactggct gcctccctga 8400 gtcactgccc acccaccagc aaataaaaaa gagtgtcatc tgctggccag tggccccatt 8460 tggccccagt gtttgcatct ttgtgctcct gggtggaagg tgagagtccg ggtagggtgg 8520 tctcacgacg acagttttct gcgccttcct ccttcgggac tccgattcct tccctgccct 8580 ttgttettcc cctcccccca tgatggttct cgctggtgac aagacccagc ccctgctcca 8640 aggacagagg cagctgcaag ccctcaggag cccccacatg gccccatcca tctagacagg 8700 tctctgatcc ggctgcaggg aggggaccct cggctgttca gctgcgactc ctccctcccc 8760 gcccgcccac tcagctgtca acactcgcct ccccgccctg gccttcccgg ggccctgcct 8820 gactgctaag tccgaggaca ccgcgcccgg agaccctcac tcagacgcaa gcaggcgatg 8880 agcaggecgge ggggcagaca ccccgcctgc ccttgagcat ccctaggcct ccttgcctaa 8940 ataagggagc cgcagctgga gcgagccagc LggCCggggg gaggggcctg cggctaggcc 9000 agggctttcc gaggggctgg gCCgLggCCg CCgCCgcagg gccccgagcc cgccgattgg 9060 gcgtgcaagg cCtggccccgg gCLgCCggEg gagggcgggc cgctcgcacc cggggatccc 9120 ctggttggaa cctcgcgctc agggacctct ctttgatcct gcagctagga ggccgcaact 9180 gctgtcagaa tgtccctctc tgtgcagctc ctatgcagag ccccacagag ggctctctct 9240 ctctccgtgt gtgtgtgtgt atgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtatgt 9300 Pagina 143

P318031NL_ST25{9969840} (1) gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgttggg gcagggggcg tgccccttct 9360 aagtttaacc ctcccttatt tcagagctct gctgctctgg ggccttcaga ttgtcttgct 9420 tttcctattt ccagggtctc ccttgactcc gggctcgtct tcgagggtcc aggcgttgcc 9480 ccggtcaccc acagtaggca ctgagcgcgt gctgtgtata ccaagccagt gctcggcgtg 9540 gtggggaacc agggcataag gacccgccgt ttcccttcaa atcaggtttc ttagtcgcta 9600 atctggggtg ggggtggaat cccttccccc atcccggccg cctacaagcc ggccatggca 9660 cctgtcctca cggtttcccc tgcctaaggg tcccttcccc ctcttctggg cgcgcgagtc 9720 ccaggggctc cttggcagag cgaggggctg gcgltggggg agcactcggc gcggctcagc 9780 caatgggaag gtcgggegeg cgeggtgggg cgggatggge gggggagagg gcggtgctgc 9840 BEECECECEE Caggcgggag BEBECCEEEA BECEEEEECC gRagCcggag BEECECECEC 9900 cgcattaacc CttCCgCggC Cgggccgagc cgcaggcgct gCCCgagccg CgggCgCtgg 9960 agcggctgtc ggcgcggtgg Caggcgggag ccaggagtcc ggggctcggg CgggCggagc 10020 gCagCgCEggEg gaCgCgECEg agCgggCccC geggetegge gggggccggc agacaggtgt 10080 gccggeggeg ggcggctcgc gctcaggcgg ctccaggccg ggcccggggt ccggagegegg 10140 gagccgggag ccgagcgagg acccggcgcc cgcagtccag tcttggccgc gcccgtgctc 10200 ggcttggeeg cggggtgecgg ggaccacgge ccggccgggc cacccgaagc ctagtgctgg 10260 gCCgggccgg gccggggtgg gLgggggccc gcccggcccg cccatgggct caggatgccg 10320 gtgcggaggg gccacgtcgc gccgcagaac accttcctgg acaccatcat ccgcaagttt 10380 gagggccaga gtgagtgggg gagggggctg agcggggagg gggctctggg ggcgggecga 10440 gctcttccga gtgtggatgg gggggcgtgc ccgggccgtg tgcgtcggca agtgcgagtc 10500 aatggggcct ggtgggcccc aggctttggc gaggggacgg cgggggcaag gcttggtgca 10560 ccggggccgt gggcaccggg ccaaagggtt tggggttggg gcttggggcg ggaaactcag 10620 ctcaggcttt tgggatcgga gtgtgtgtga cacgctgagt gtgcagggaa gggatgtggg 10680 atggagtgtg caggtgtccc caactctggg tgcgtggacg tggatggggc tgtgtgcaag 10740 ggcataagag tgtgtacgac ccaaaccctg ggatcccgag agtccagcgc gcacactggt 10800 cttgccagat cttgactgtg ttgtgccgga gggactgaaa agccgtggat gcagggtcag 10860 Pagina 144

P318031NL_ST25{9969840} (1) ctcataatct cttcttgtgt gtgccgtgtg tatataaccc tgagcccatg tgtcacccgt 10920 gagttagacc ctgtatgggc ggtggggagg tgtctttgac aaggctgcga gcttgtgggc 10980 tctgtggaga cagggtgtcc tggggctaga gctgggcttt gagggtaagc agctccctgt 11040 ggtaccccca agttgtgggt tttcttgccc tggagactgt cacctaattc ccttatgggg 11100 accgggagta cagcacccct aatgcctgcc tggtagggag gcttctagaa gggtcccaga 11160 ttaggcaaat gtcctggggg tgggcagcat tcatgtgcct gtccctaagg ctgcatgagc 11220 cacagtgggt gtgactgcca agggccctgg gagttgggag aggatgacat tccttgtcca 11280 tgaccctgtg agcacaggac aacgcgtgct tgtaacggtg gttgggggaa ggaggggtca 11340 gtccgatggt cagtattgtg tgaagcggag gaacaagatg atcctgaggg ggtggagaca 11400 atgacactgg ttgagggcct ccctgagagg tgggggtggg cccaggggga ggctgtgggc 11460 gggcctacga ggaggctggg atggatgtgg gggggggtgc tggattctag ccctaccttt 11520 gccactgact gccatgtgac cttgagtgac tcttccctct ttgggtctca ggttccaggt 11580 ctgtggagga ggggcaggct ttctgtccca tccctgtatt ccaggatctc agtgaagggt 11640 gtgtggtggg gtggggcatg ccttcgaaga aacttgctct tctttcccca gcctgggaga 11700 gaggacaagg ggtggttttt cttctatgaa gctggggtgt gtggctgggc ctctggactg 11760 ccttggactg tggcagagct ggaggccgag ttagggggtc ttttcaggtg acaggaaacc 11820 ctgactcctg gaattgatgg ctgcctggga ggtctggggg gccttgggaa ctggccaaaa 11880 gatgaaccag ttccaaaagg caactttctg ggggccaggg gaagggagaa tcgagaaagg 11940 tgggaggagg ccaagtcctg atggcctggg cctatctcag ggtgccctgc tgcagcccag 12000 ctttgcacgt aggattgcct agggctgcaa cctgagggct atgcttgctg gggaggggag 12060 gggaagggag ggggagaatt ccaactgcta ctgaaagccc cagcattaca ggccaaagtg 12120 gccggaaact tctgtttata aaaagctctt atcccaggag catcttgcct ggcacaagtg 12180 aggctgctgc ctgcctgctg ctcactcgcc agcctggctc tccgggcagg cctggagtca 12240 gggagccaag tctggcagaa atggaaccat gggggtgctc atccaggatg aggccaaact 12300 gaggaaggcc tcacatccct gggcaggtcc cctttcttgg gtctgttttc ctacaagtgg 12360 ggccaaagtc tcaggccccg cccaggctcc aggtgtgtcc agggtttgga gctgcaggtc 12420 Pagina 145

P318031NL_ST25{9969840} (1) cctctctttg cttcctatga ccttgggctt ctgcctgtga ccctcagctt attcttctgc 12480 ctctgccaga ccaaggttca aaagctggct ctatcattta caagctgcaa gacttgggca 12540 tggtgccgaa cttctctgga gccttggttt cttcatctat aaaaggagga taataacacc 12600 taccttatgg ggttgtcaga ggacctaatg agatgcagcg tccttaggtg ttcaacgtgt 12660 aacttccctc atcctctctg gcacacccac ccctcagctc ctgactgccc ttggggeges 12720 ggtggtgtge agaagttcta gggggtactg acctggtagg atcctcagat aatcggggag 12780 tgatttccta gggagcctca ggataaggag tggctcaggg agcgcccccc agtctggaga 12840 gcagggaaat taaaagcaga atccagagag ccgatgcaaa gggaatcctg gccgccatct 12900 ctggectttc ccaggctccc tttctaggcc cagtgtttat ccctccgccg cggctgctga 12960 cacgcgttgc tgacacccgc caggctggcc tcagcctcgg gcatggagga ggCggCgggc 13020 tgctggcggc CLgCCCggEEg gaagccacge agcCCCgCCCg CggECCagEC gEaggaggEEg 13080 gcgegeegeg cggcccggcc ggccaggcac ctgtatcggg gcacacgcac agctgcgttc 13140 gccccctgct gggaccccag tcccagccat gcttcctgtg gcggggagtc CCCggEgCggC 13200 tgtgtgagtg gagaatgtgg ggaagggctg ggagtcccgg aggctggggt ccccgctcac 13260 gcgcactctc ctccccgccc ccctaggccg taagttcatc atcgccaacg ctcgggtgga 13320 gaactgcgcc gtcatctact gcaacgacgg cttctgcgag ctgtgcggct actcgcgggc 13380 cgaggtgatg cagcgaccct gcacctgcga cttcctgcac gggCCgcgca CgCagcgccg 13440 cgctgccgcg cagatcgcgc aggcactgct gggcgccgag gagcgcaaag tggaaatcgc 13500 cttctaccgg aaagatggta ggagcgggcc gggECgggEC cacgaccagg ggcggggtca 13560 agaggggecgg gaccacggeg aggggcaggg LgCgtggggg TgLggSggtg TggEgECgEgc 13620 tggagaacgc cccgagcccg ggcagggttc tgtgggggtg tgacgeggge gggggccctg 13680 cggtgtgcct gtgggaggcg cggttccggg gcctgcgcct cactctgctg agatggtttt 13740 caggagggca aaggagttgc taggctgtgg gtctggctcc tctggtcctt tttttccctg 13800 ggcaggtgtg ggcacgtaga gcaagttgga ctagagttag accgagctgg gatggcaggt 13860 acgtgcgtgg atggaggtta aaggtcagat tggcagttgt gcgctgacat ccgggcacag 13920 gcccctatct accttagggc agttgctgcc aggtgacagg tctgggagat gtcatggagg 13980 Pagina 146

P318031NL_ST25{9969840} (1) gacaggaggg ctgcaggact ctggctgtct ctgctgcccc cctcctgccc tgcgcttcag 14040 cagcacctgg gggaggtgcg tgcctctccc cgcaacctca gttccctttg tctcagctgt 14100 cgagcgcccc gccctgccac cctctcctgc gccaggtgtc tttactcagc caccgcctcc 14160 cagcacagct gctctgggct tccggagagc ggataaacag agcccggggt ggggctgctc 14220 ccacaccgtg aatgccctgt tggggtgggc tctggtcagg gcgggcagag caggaggact 14280 geggtcctgt gggcagaatc cggctgcaga ggcggctctg gaacgcgtgc aaaccttgct 14340 gcttcctagc gtgtgtccca taagcattga ctcaaccctt gtgagcctgg caggcactag 14400 gctcttggag atgggcagga cagatgaggc tcctgccctc agggagcttg tctgctgtag 14460 acagtgagca cgctctgctt tctgtttggg accaggccag ccagccccat gtgccagccc 14520 tgtgtgctca gggttggctg gggtggccct gcaaagcttt gcatcctcca ggctgtgctt 14580 ttgcaggcgt gcacagtctg tggcatcact ggacggcact agagggggca tggtccctgc 14640 accctgtgga agaaggttcg ctcacagcaa atcagggttc taactggcag gaggcagggs 14700 ctgtgagact agactcaaac cctgaaggca gtggatggtt tctaggggct agacactggg 14760 ccaaatgctt tacttctctt atctcaggca ctcttcacaa cccagggagg tagacactac 14820 tgttgtcttc attttggggg atgaagaaac tgaagttcac acagtcacac agctagaaaa 14880 tagcagttgg gaacggagcc cacatctgtc tgaccggagc cctgtgcgct ctgttctgtc 14940 tgtcatatat tgaaaatgac accaggtagt tgcaggtcta tctgtatgct gtacattatc 15000 tcatgtgaaa ttttattgta aaagtttgcc aaccacaggt gtactgaaaa gaatatgaac 15060 ccataagtcc tgggtttgtg tctaagacaa tgctggaaat tatacactac tggacaagtt 15120 ggttgacttc cctgggcctg tttctacttc tgggataacg tgccttgctt tggaccttca 15180 caagggtgct gagctttcat agagagtgcc tgcaacgggc cgtgctctgt aaacacggtg 15240 ccagtcccac tacgatgtag ctgtgctggg gatttggcct cttggcagca gcggctgagg 15300 gctctttcct ttggctgtgg tatgtgtgga gtgctcttag gtggagctgg ccctccctcc 15360 cagtctaggc tggccccttc tggagaggat ggctatgggc ccccttctgc agaggagtta 15420 ttgtgggatc acctaatcca acccattcga cttgcaggtg gaaggacgga gaagcagagg 15480 tctgatgacc agtgaatcag tggaaggagt gggattagaa cccagatccc cagcccaagt 15540 Pagina 147

P318031NL_ST25{9969840} (1) tcaaacttgt cccctctcca ggttacaact ccaaaaaatg gggctgtgac ccagaaaact 15600 ctgtgccagc aatctctgtc tgtccccttc ttcactctca cctagtttcc acggggctca 15660 ggtgtctcct gttggccaac aggctggcct ggcactcagc cacctaggac cccatgtatc 15720 ctggcattct aggcctgggg tgatgcagac agatttcctc agcctccgct tcttctgcct 15780 tgaaacactc agtccctcct gtgatccccg ggacctggct tgggctgcgt gtgtgcgtgc 15840 ctgtgtgtgt actcacacat gggcctggta gggaggcgga ggtgaggaat gcctgcgcca 15900 ccccttgttt ctgtccaggc ctttctcagg gtctctgcct gggccagcct cgggtagggs 15960 gagctgggca ggaggtggag ccttctgctg gcaggcgccg ccgtggacag ctgtgctgtg 16020 ttggtgcctc tagccttgct ctgcgcagtg tcagggcctg gagccagaca gccccattat 16080 ttagttgctg ggactgctgg gaggtgggga ggctagactg ggggagtgtg ggctgaaggg 16140 agagacatga ggtgcctggg gctcactggc cctgtgcccc ttgaagtcag gcccgggatt 16200 caggtggaat tgggggcaga gtccagtggt gtgaggctgg ctggcggtaa catccccgca 16260 aggccctggg gaggcacccc gggcagcagg tgctggctgg gtccacacct gtgctcccac 16320 atcgccatgg gggctgtggc tcctcctctg ccagtcctca cagagtccct tctctctgtc 16380 cccctgcatc ctctctgctc tttgttcctg cctctgctcc ctgcttccct gccaccgtgc 16440 tcagccagcc tgactgtgcc caccttctcg gctcctcetct cactccccac atctccttgc 16500 ctttgtcttc tgcctgcctg gcttactctc tgtcccttgg tctttatctc ctctctggtg 16560 cctcctcctg tcttcettccc tegcctcagc accccggaac cttgctcctt gcctctctcc 16620 ctctctccca ttgtccccca cccctcggag cctctatctg gaaggctctg tctggctgct 16680 catacctctg actggcctgg cctggctcca tgtccctgtg tccccgggag cttgtgcgtg 16740 ggtggggacg gtggtgtctc tggagaggag tctattagct tctccactcc agctgcaggc 16800 cagttgccat ggtaacaggg tcagagggcc ccccactgca acagctgatg ctgcggctgt 16860 tgggaggggg tggggacctg ctgtccaggc ctgctcccca gtgcactgtg cccagggcct 16920 cagaggtggc cccacgccca gtgggcttcg gtctgtcccc ggccttggaa cccagcctcc 16980 ccagtgctcc tgcaggcctc agacctcaga ctgccttgaa atccttagta cctcctgtga 17040 tcagccccac tcctctgggc ctcctgccct tctcegtccc ccaaggccgt ctgtcctgtc 17100 Pagina 148

P318031NL_ST25{9969840} (1) ttettecttg ccttcttcat ttcttecctgt gcccattcct ttaaatcaga cgccctgctc 17160 ttgtcaccac cacgcctcac cttctccctc atggctgacc cctccccagc tcagacaccc 17220 actttctgct tcttttcagc cccctgggtc tgagctgagg atgaaagaag cttttccttg 17280 aaggggagcc ttgaggctgg gaaggcaggt gggacaggga ttggtgtata agtgggtttt 17340 cttgtcttga gataggaggc agaccagagc gatcccagag gatggggctt agacacagga 17400 gaacttttcc tgtggcactg aagctatcag gggagcgccc ttcatcatgg ggacagcggt 17460 gttcctatcc ttctggtcaa ggcctggtgc agcctggaga cagggctgag tgggagaatc 17520 tgcagggctg ccaggtgcgg gcgggggtcc atgggggccc tgcttccgag ccagttgcct 17580 cttgccctgc agcaccttcc ctccaccttt cccttctcta gctggatcag aggcctgttg 17640 gaggggctcc acgggggtca ttcctgagct gcggctgtct gtcctggtct gccggggctg 17700 cagtcttcct atcccagtcc tgctccaggg gtgaggcagg catgacgcaa ctgaagggtg 17760 ctatctactc cttgacccac cctggggtct ggagagcgtc gggagagcgg cttctccgtg 17820 ttgcgtcttc tgcagccccc ggagaggagc tatgtaaata tttgcgaaga gctgtctgag 17880 atgggagcag agtttgatgg tggccttggg ctctggggag gggggccatg gggcagcaga 17940 agggccagct tgggggtgtg tgagggggga gactgggcct cttttctccc cagaggtggc 18000 ttcctgggtg gggtgatttt ggaggttcca agaaagccct tcacatgttg gtatttccct 18060 gaagcgttcc tgaaactcct cccttagaga acttctgcgt ttagagtgct cctttttect 18120 taaaaacacg ttctctctca ttgccccctc ccagtataga gacagaaagt atttagccct 18180 ttagtgtgag ggagtttaac agaacttccc tcccgacgtg ggcaggaggg ccatgggagt 18240 cagggtcagt agaagaggga ggggtgctgg gacaggtgat gaatgtgggg tgctccccta 18300 aagtcccctc catgtcctta gtcttgccca taaagatctc tatgctgtgg cctgtggttg 18360 gcctcagata aacccctctc ctttttgggt gcttatccag gatgagaaac accttgggag 18420 ccaggcagcc ttggtcctcc gcctcctgca gtactggagg aggttccctg tccctctccc 18480 cgccaccagg ccttgagctc caggtgggtg gagagcagcc catctgcttt gttctctgtt 18540 gtatctgcgt gctcagtgca cagcgagagc tcggcactgt ttgctgaatg aatgaatgaa 18600 tggggtgggc agtgtgctgc gttgcaggag ccttccctgg gagtcaggtg acttggcagg 18660 Pagina 149

P318031NL_ST25{9969840} (1) agtttccagc ttggttacag acttgctgtg tctgggagtc ctctagcctt gctttgcgtg 18720 gacattgtgg ggcacaggag catctggggg ctcccagctt caccctcaga ggtggaccga 18780 ggaggatgge gtggggggac agagtcttag agctgccaca gagagcacag ctgaggtggg 18840 ggtgatgcca cctgaccagc agcacaaggc acttctattg ttgtgactgc actttgtagt 18900 ttgcaaactg gggaaccatg ctttaccgca ggtggtcctc atggccctgt gaaaggcagg 18960 attcctctcc gtcactttat gggagggaag ctcttgctta gaaaggctga ggcacttgcc 19020 caagtttacc cagctggcgg gtggcctgag tgggaacccg agcctagctg tcccctccct 19080 gcccagcgtg gaatgccact gcaccagctc tcttgccccc agagagccct tctggggcat 19140 gatgggctca gggcctcagg aaacttggat tttagctggg gtctgcaaac ctccattcct 19200 cgtttgctaa gtgggatcat agcgcctgtc cctgatgcac tttggggtgt gagggtgttc 19260 ctggaatgag atgacagaca cccacaaagc agcgtgcggt gccagagctc attctcctcg 19320 ccatgaagca gggcgtcacc taagtggctg cagctcctag cccgtgctgt tgcaatcgtg 19380 ggaaatgagc ctgatccggg gcctcagcac ccctgctcct atggtgcacc ccgccccatc 19440 ctgcatgctg cctgcctctc cccttccctec cctgcaggct gctgctggcc ccgggtcctg 19500 cctctccccc aaataattga aaccacagca tttgtgttat tgctttttce tttctcttte 19560 atttttttce tttteccttt tatctcactc tccagtctca ttaccctccc cataggcaac 19620 cgttcaaatg tacttactgt gaatgtgttg cttgcatatg ttcttgcaaa tgtgtatttt 19680 gtctggatat atgcatatag atttaattta tgtaaatgat attgtgctat atgttgtatt 19740 ttggtgttcg cttttcttac ttgggaatgt gctttaagtc cagctgggtt gctgttcgac 19800 atctagtctg taggtgcgta ttcagtggca tgcggccgcc acgcttcacc cacccactct 19860 cccggggagg gaccccacaa tgtctccaac tcccagccac tgtaaatgag gctgtagtga 19920 atatccttgg acatggtccc ttctggactt gggtgaggat gtctgtggga tacacgcccg 19980 ggagtggaat tgctggatcg cgggacatgt ggacacttaa ctaagtactg ccatgcctgt 20040 catcaccctc cttctcagag ctcgagattc ccatacccgc atatcttggc cactctcgac 20100 attatctacc tttctaaatt ttgccaatct aataggtgac agtgatttct tgttgcttta 20160 atttgcattc ctttgatttc tgttgagtct gagcatctct taggcctggt agcctcttgg 20220 Pagina 150

P318031NL_ST25{9969840} (1) gcttactttc ccgtgaattg cctgctcatg tcctttgctc attttaattt tgacgtcgcc 20280 gtcttttgtc cttgttgatt tgcaggagtt gcttgtgtac tctagagata ccaatgcata 20340 ctgttaaaca ttcagtgtat tccagatttt aattccttgt tgaactggaa acagatattg 20400 caaatatctt ctcccattca gtctctcttc aatttatcca acaaagatac ttaattttga 20460 tgcaaacaaa ttcattgatt tttagctttg tttgtacttt tgaagtgtct caggctgcag 20520 agggtctcca ttttcttctg ttctcttaat tgttttgctg ttctcagtta ggtctataat 20580 ctatcctgag actaccccgt atgtggtgtc aagtagggat cgagttttat tttttccatg 20640 taatgtgcca actttcccaa cgcatcttgt aaacagtctg ttcttttccg ctgaattgtg 20700 gggcagcctt tatcaggtta atctcccaca tagccatgag tctgtccctg tgctctctgt 20760 tctgttccaa atatttattt actggctctt gcattgatat agcttaacaa aaacctatag 20820 ccttccgtat gtcttagcgt ctgctgaggc aaatcctctc tcttcattct gttttttaaa 20880 ggctgacata gctatttgtg gatctccctt gttcttcata aactttattt attaattaat 20940 taattttttc agatggagtc ttactctgtt gcccaggctg gagtgcagtg gcatgatctc 21000 ggctcactgc agcctctgcc tcttgggttc aagcgattct cctgtctcag cctcccgagt 21060 agctgggatt acaggtgtgc accaccacat ccggctaatt tttgtatttt tagtagagac 21120 ggggtttctc catgttggcc aggatggtct cgaactcctg acctcaggtg atccgcccgc 21180 cttggcctcc caaagtgctg gattacagac atgagccacc gcgcctggcc tcccttttte 21240 ttcataaact ttagagaaag tttattgaat tcttggaaag aaaacaccca ctagattctt 21300 gattggaatt agtttggttc tatagattaa tttggggaga actgacatct ttatatttgt 21360 cccactcaag agcctggatt gtctctccat ttaccagatg agttctgtat ttcttgctag 21420 tttaaggttt tgttcccttt tgccccgact ccaacagggt tcttgtgcct tcttggtcaa 21480 ttcctggaca cttaagagtt tttgttgcta ttgtaaaaga cgtattgttt ttgactatgt 21540 ttcctaattg cttgtgtaga aaaatgcttt atatttttgc gggctgatct tgtatcttgc 21600 aaccttgctg aactctctga tctaattagt tctaattgtt tatctgttga tttcttgggg 21660 gtctctaggc aagtgacccc gtcatctgca aataatgcca gttttcgctg tttatgtttg 21720 gatcaccgtc acatgttttg atgctcgaat gttccacctg actttctggg ggcttttcat 21780 Pagina 151

P318031NL_ST25{9969840} (1) tagcactcgt atttgtttgt tggtgcttca cagtgtcctg tgtgtgtgtg tgtgtgggee 21840 BEZEEEaggEg ggcaaatcaa tgaggaatta taactcttgg tttaagctgt gactcatggc 21900 agagctggaa ttgggacagg gctttatcaa agcttaccag gctgtctggg gtatgctagg 21960 agccaagaca gagcacagac ctagaagcct acaggccact cactagagga gaccccagag 22020 tgtcccctgg tggacagtgg cagctgtgtg caccttcact gccctgtggc aagctgtgtg 22080 tggggtcaca gtgaaggggg gctgaagggg actgaggctc agaagtctca cacctggtgc 22140 agttcctgct tttgccgcct tcccttgggt tcctgggtgc tcagcctctg tgttccatga 22200 aaactgatga ccaggttgaa ttttgcttca tcaaaaggca ccttaattcc catggttgct 22260 ccatctgctg tccagacttg ggctggaggg caccacgggc ttgaaatggg caaattctgc 22320 tgtgaaaggg tgggctgtgc ccctggatct ggaagggagc tgagagggtg gggacatggc 22380 ctgtccccac ctgttatgac tgcagatcac cacccctgag tccaccctca gttcttctgc 22440 aactgccagt cctgccctgg cctcagggct ctgactcgga tggacgggga agcccggcca 22500 gtgtacagtg gccttctaga cgatgggcat tgtttggaca agttctccaa gagcagcgtc 22560 catgtgggtc agacggagca gtcaaggaaa cctttctaga gggggtccta gaaggctggg 22620 gaggagttgg agggaagagg gStgggggcc atggggagca ggagggaagg gctgaagctg 22680 gggcgcaact gcccgggact cacgcgcctg tgcagggagc tgtggagagc tgcttcctgg 22740 catcagcgtg cccttgtttc accttcccag gcttctcagc aggagggcag cgggtgggac 22800 ttccagttcg tgcaccaccc gagaggcaga gccgggcccc agagctttag agaagctaga 22860 ggacagagat cttctgaggc attgctgtgg tccggggcag agatggctca ggcatgccac 22920 atgcagtgga tttgggtggg tctagatgac tacagcggcc cccaagaccc tataaatgat 22980 cccatggatt gctggctggg tctcaggctg gaaggggtgg tggaggagtc tgtctggaga 23040 tgctgcttct gtggggtcag ggtctgagct tggggcccct caacctgctg catttcccag 23100 atcctggagg caggagctta ggactggtct ctctctcatt cagcacacac acacacacgc 23160 gcgcacacac acactctctc cctgtctctg tctctctccc tecttectctec tcattcactg 23220 gttggcctct tgggctccct aactctgtcc cctgggcctg caatgtccct ggcctttcect 23280 aggcccctgg ctgcccatga gagctgtgtt agtgacacgg tggccccctg CCaggagcgg 23340 Pagina 152

P318031NL_ST25{9969840} (1) ctcctgctgc atcttcctgt cgccgccacc aggctaaata taacctgaca ctgcacagcc 23400 ctgggctgct gctccggaga cagcctctct gecectggggge ggacaggccg actggagccc 23460 cagcctcctg ccctcagcgt gactttgggc ctgtcctggg cacagctcct cctgctccga 23520 gcgaggcagg gaggcgacta gggcgaggat ggctgcctcc ctgaggtcac cttccctgca 23580 gggacccagc accccgcctt gttagaattc tggatgcttc cacaaagcca tctgtcatct 23640 catctggcac ccagtgcagg aatcacttgt ccccacagta cccgcaggga gcctgaagcc 23700 cagggaggtg ggtgacctgc ccatagccac agaaaataga gggaacgcag gagccaccat 23760 ggctggggta gagtggctcg gctccctggg gctcctccac ctgtctggct tgttccectct 23820 ccttccctct tgcccatgca tcaaactggg gtcctgttgg atgggtggtt cactgggggc 23880 aggtgccagg cgtgtcttgt tggtcacggt ggctcaacac ttcgcaggca cggatgtgct 23940 tagtgatttg cagaggactg ggaggagctg cctgggaaga agctgtgata gcacctcatt 24000 tacggaggag cgcacggaga ctctgacccc agaaggtgcc taaggtcact cagggccatc 24060 tgcagcctct tcctccccac ctggtgctga catgggagat aaggcctgcc cctgccacag 24120 ccaaatgtgg ctcctgcctg gggcctggcc taggctagcg gtgcccagag gtggggaatt 24180 gegtggagcc cctgacctcg gatgggttgg gatgctgggc aggaacagtg ccccccaccc 24240 caggtagact gctgggtgtg gattggcacc aaatgaaagc aagccggcgt tgtgttctga 24300 gaagtgatgg acaccttctg tggtagcagg atggcgctgt acctgccact ctgggaatgt 24360 gtccaggcct ggctctccecg ctctgctctg agctgcatgg cctctggagg cctgctaggt 24420 ggccagcctg cctgcctcac ccaggccatt gggcccaaag gcacgggggt tctaaggagc 24480 catggagcct gtgcagggat ggttgcgtcc atgggtgcct cccctttcag agggcccagg 24540 aagcagaact gggccaggca gggagaacct tctcacccaa agtaaggtgt cttcagccag 24600 cgctggggct gaggctgggg tgggagtggg gtcggggctg gggctggggt gggaatggEg 24660 ccagggcaga gtgttctggc attagccaca tgagccaggc tggccctggg gcctctcatt 24720 gctaagctcc ctccaccctt gtctgcctct cttctgtctc aagcttatgg tgacctcaag 24780 ccaggctctg agcttcacat atcttaggcc atttcacccc tacggcccta gggggtgggc 24840 aggatcaccc tgtgttgcag atgagcgccc cggggctctg ggccggtaag tgaccggccc 24900 Pagina 153

P318031NL_ST25{9969840} (1) aaagtcattc taatgcaatg tgcagaggcc acctgactcc acagcccatg ccccctccct 24960 ttgagactgg gaaccctctc ccctgttgca ccccctccgc cctccttgtc tgtcctggct 25020 gccccctgag tgcgcccagt gctggctgcc tcgggagtgt gggctgtgcc tgccacggag 25080 tggctctcag cggaactgca gcctgcagca gctgggtcct tcgtctctgc cctggggagc 25140 tggggccact ggcaggagcc agggcgagtc ggtgggagaa gggagcagag cgagggcctg 25200 ggggtctgga gtaggtggca ctggggcaag tgaagggact cccatccccc cataacccag 25260 ccagtggtgg gatggagtct ggctgggagg cctggctgaa gagctgggag caggtgatgt 25320 ggcagttcac cgaagcccca gccgacgcgg cagggtggtt gcaggcagtg cctctgagag 25380 cccaggccta tctggggccc ctactttcct tccttccaca tccccttgat gatcgctaga 25440 tctcccccgc cccagcccct gccatcaggc agggctccag ttcacatttt ttgaataacc 25500 aggcctttga cagggttatt ttgaaaggaa aaatttcttg aatctcgaca aaggaagagg 25560 gccagcagtc actcctctct ctctctcectet ctectctectet gtctctcgtg tgtgtgtgta 25620 agtgtgagtt acaggcttgt gctgtgaggt ggcgtatacc tagagtttct gcgaccctgg 25680 caggggattt tgagatgtgg ggtcctggac tgggtgggct tcagaatgcc attctgggct 25740 gtcttgctga gcagcttgag ctgaagaggg gacgggctgc tgaggtctca aggttggaat 25800 ttctggggcc ctgtgttggg gacagtctcc agcccattct gaagtttgag tccatcttgg 25860 ctccagactc tagacttctc tgaggcaagc tccctggtgg aggacttagg ttcgagggcc 25920 tggctggctg tggtccctgg ggtgggtgct ggggtctctt gggctaaggc ttcctggtct 25980 ccagcctctc ctcagtcctg gtgaggcgca tccctgttca ggccgctgcc ggccatcacc 26040 atattagggc ataaactcca ggcaggctgt ctgggtgcca aatggtctcc agctgggccc 26100 agccccctgc agcttccaac accactgctg tgtgtcagag gccagaggtc aggggacaag 26160 agtggctgct gcagggagac ccagacagag ccccagagct cagggccctc gggcggggct 26220 gtgagggtct gggatgtggt gagtggaaga gctgtggggt ggggcggaga gttcctggcc 26280 agccccttca cctagtgcca tcctgtttaa gctggtttca gaagacctcg ttcccctaga 26340 tgaatcccca gcgggttaga atccccgagc ctggccagca tctgccatgc atcaggccct 26400 tcccacctgg cctgctccct gcctccccag cacgcagccc cttgacctca ctgtcaggct 26460 Pagina 154

P318031NL_ST25{9969840} (1) tcaagcacca gctccaagtc ccaggggtgg cgtcgcctcc aggttgccgg tgtctgttgc 26520 cggcttctac cttgactcac agcaccacca ctgtcgcccc tgctgtgggt ttgagatcag 26580 gagtaggggt gagccaggga ggctctagga ggtagaaagg tgaggagagg ctgggtgegg 26640 tggctcacac ctgtaatcct tgcactttgg gaggccgagg caggcagatc acttgaggcc 26700 aggagtttga gaccagcctg gccaacacag tgaaaaccct gtctctatca aaatatacaa 26760 aagttagctg ggtgtggtag catgcagctg tagtcccagc tacttgggag gctgaggcag 26820 gagaatcact tggacctggg aggcagaggt tgcagtgagc caagatcatg ccgctgcact 26880 ccaccctggg tgacagagtg aaaccctgtc tcagaaaaaa aaaaaaaaaa aaagtgaggc 26940 tgggttgcta agatggagtg ggactgcatg gtttgtcacc cttgcctctc ccccatgcct 27000 gcagcctccc tgtcagcatg gcctgtcact gacaccacca gccagtgctg geccccagggc 27060 gtatgactct aggtgctagg cactaacgtg gggacgggag acactgggag gcagagaaga 27120 cagctgagca gagggggagc tgagaggaag gaagaggctc cctagggaga gtgggagatg 27180 aggagcctgc ctggtgggtc cctggggggc catagaggag ctggagcggg ctgttaactg 27240 ccttctggtt tcattaagga cttcctgtgg gcaggaggtt ctgagctggg ccttaggtgg 27300 aactgagtgc ttggcctaaa aggggctcct tcagaaatca gcacgggttg gggggceges 27360 gBECggLgEggg cagcaggggg cccatgtgct caacagggag ctgagggaga ccaggggtgc 27420 ttggcttagg ggactgacct catctggtac agggcctttg gtggacagag agagactgga 27480 aaagaaaaag tcaaggatcg aggggaacgc gaggcgaggt gaagatgaaa gatctagaag 27540 cagtgcttct tagactcttt ggtgaaggat cagtgttttt taatgctaat atgttatagg 27600 ctggtacatc tgtgaaatac aagagcacat gcgtggattt tgtggtgatg ccaaattgct 27660 gtaaagtttt tgaaattctt gctctcaacg tctgtcctta tcttgtgtgc tgacacaacg 27720 gctggtgcta atctagagag aacagagaga cagatagtaa cagctaaccg tctgtagtgt 27780 gcggcatacg ccaggcattg cctaagcgct cagcatgtgc tgacttggtt aatctttata 27840 actcatgaga aaagaattat atacattatg tgtatcacaa catcactgtg taccccataa 27900 atatgtacaa ttattgtgtc aattaaaaag ttaaaaaatt ttaaaaagaa aaaattatta 27960 ttcttcccat tttatagatg gaaaaaatga ggcacaagga tgttaaataa tttaggaaaa 28020 Pagina 155

P318031NL_ST25{9969840} (1) aagcataagt gattaagcag cagatccggg ctatgaaccc agagatcgta ggcctaaagt 28080 ctgtgggctt aagcactgtg ctacagcgcc tcgcttgagg gaaaggcaga ggtgctggga 28140 ggatatgagg aaatgagata gggaggaaat gagatcaaag atggcagaag aaaggatcat 28200 agccagcgtg agaaaaagtg cagccgggaa agagctagat aaatcatata ggcagagaga 28260 geggtagggg agcctggcag caacaagctg gggtagacag attgagggga gccataaggg 28320 cggcaggcac atggccgggt gggggatcag gacgggagat cccggagagg aagggccata 28380 Cggggaggca gaagtggacg ggcccacttg ggttccaggg tccatcctgc gtggctttct 28440 gctctgccca ctgagtgggt gccaaggggg ctatgtcctc ccactctgca gggagctgct 28500 tcctatgtct ggtggatgtg gtgcccgtga agaacgagga tggggctgtc atcatgttca 28560 tcctcaattt cgaggtggtg atggagaagg acatggtggg gtccccggct catgacacca 28620 accaccgggg cccccccacc agctggctgg ccccaggtaa gtgtacttgc tttagggcag 28680 gctcaggggt tcgtggtctc atttctttgg ggctgtgagc tgtccaaggt caacgctctg 28740 caggaaggga ggatgtaatg gttgggtggg gggtcccccc tttggaggtg ggaagtgaac 28800 cctgtaccct gaggatgtgg cttggttccc ctggcccagg atggacacat aggggtggaa 28860 tgacagggcc cagaatcaca ggtccttggc gtgggctagc accgtttccc tagcttcatc 28920 tgttgggctt cccatcccag catcccctgg gagcggtcct ctctccagcc tggcttgagc 28980 agcccagggt ggggaacatg accacctggg agcagctcct cccctcagta ggaagctgtg 29040 tcctatggac tcagctgtgt gtagtttctt tcatctgctc cttgttcatg gactggccaa 29100 gtttcctggg ggtctgggct agctgaggag aggctttgag ggaatgccca cctgggctgg 29160 accatcccct tgacaacaaa ggctcggagc cccttcctct gtcaggatgc cctctttggc 29220 ttaattttaa tctttccctc agcatcatga caaaagtgct cacctcttgg tttcttacac 29280 tctaaaatga cagtgtcagt aaaactggcc ctacctgctc atcggatgcc acccctgacg 29340 gcagggcccc tggcattgtg tcgcccactt aggcgcaggc cccacgcact ggctgtgtga 29400 cctccagaga gtgacatgac gcctttgtgt ccacacttcc tcatgtctaa aatggggacg 29460 ctggcaccca gcggccggtg tgagagacag ggatgattta tggaatccgc cctgaagtgt 29520 cacttcagat atggcggttt atgatgactg gattctagaa tgatttttga gactcgaggt 29580 Pagina 156

P318031NL_ST25{9969840} (1) caaaatattg tgaatggaag tggggatggg gggttgcccg ttcccctcct tcccttaccc 29640 agcggctggg ggtccatttc ccaggccttg cccgctccgg ggctgctcgc gatccccacg 29700 accacgtgcc tctcctctcc cgcaggccgc gccaagacct tccgcctgaa gctgcccgcg 29760 ctgctggcgc tgacggcccg ggagtcgtcg gLgcggtcgg BCggCgCggEg CgECEgCgEgEgC 29820 gccccggggg ccgtggtggt ggacgtggac ctgacgcccg cggcacccag cagcgagtcg 29880 ctggccctgg acgaagtgac agccatggac aaccacgtgg Cagggctcgg gCCCgCggag 29940 gagcggcgtg cgctggtggg tcccggctct CCgCCCCgCa gCgCgcccgg ccagctccca 30000 tCgCCCCggg cgcacagcct caaccccgac gcctcgggct ccagctgcag CCtggcccgg 30060 acgcgctccc gagaaagctg cgccagcgtg cgccgcgcct cgtcggccga cgacatcgag 30120 gccatgcgcg ccggggtgct gCCCCCgCCa ccgegecacg ccagcaccgg Lgagggcgcc 30180 gcgggacccg ttccgcccca gcccacgctt cttctgctgc caggcttgct gcattctggg 30240 ccctcggctt ctgacctttt cactacgtcc tgggtgcctg ggagatggaa atcccattaa 30300 gcacttattt aattccatta agtggcttgt tgcgctatta aatgtccatt catcccatta 30360 cattcttcca ctcccagagc cctggtgggc ccctggagct ggctgtcccg ggacggcctg 30420 gagcagggat gccctgggca ggagacgtgc ccagcctccg aacaccacct ggccccctgc 30480 ctgccagcgt cctggagcag accgtttcct cttcctcttec ctggtctcag ggctgtctcc 30540 tggctgcttc cttagagtgg gacaggggtc tgacctctct tgtttcttgt gactcccctg 30600 gtctcttgag ctgaccccat ggactcctga ctgtgcaggg cctggtctcc actctcgatc 30660 tatgggtggc ctgcctggcc tggggccctg ggctgctggc ctgaccacgc tgcctctcca 30720 cctaggggcc atgcacccac tgcgcagcgg cttgctcaac tccacctcgg actccgacct 30780 cgtgcgctac cgcaccatta gcaagattcc ccaaatcacc ctcaactttg tggacctcaa 30840 gggcgacccc ttcttggctt cgcccaccag tgaccgtgag atcatagcac ctaagataaa 30900 ggagcgaacc cacaatgtca ctgagaaggt cacccaggta ggcgcccagc ggccgggctc 30960 tcctctcacc ttggaggagc ttggagaggg ggtgggagtg ggctgtgatc cagagccggg 31020 tggagggggc tagccagtga gggcctcttg gagggaggca gacctgaggg ttggggttgg 31080 caatctattc cagagctgcg atggggggag aggggacagt ctgagaagtc ctggagaggg 31140 Pagina 157

P318031NL_ST25{9969840} (1) gactggagag gtctggtaaa gggctgggaa tggcagagcg gaggtcgggt ggatggggtg 31200 cgtgccttcg gggcacaggg tgaggacagg gcagttgagg ctggggacat gacagggcca 31260 gtagcaaggg gggaaggcgg cctgagggac atgaaaggtc tggctgcagg gtgtgtgaag 31320 gaagtactag gggccaagcc aactgggagg cttcaaatga gaaggggtca cagaggtcaa 31380 ggacaggagg gtcctaattc ttccaggcca aggtgatgtg ccaggcagag ggtgggcctc 31440 agtcctgtgg ggatggccag gatggcctcg aggggcccaa ctatgtccct aaagcatggg 31500 agaagcaagt tttcttctca tagggaggtg gtgggtacaa agatatggaa gggtcaggtc 31560 cctgaatcct gggccagaga agacccctca gccttggggg cattagccag cctggtcttc 31620 tgtcccctcc tgggtttggg gtcctcctgg cagcaagagg acctgcgacc cctccatggc 31680 ttcatccacc cgacacctta tcctagcctt tgggggctgg gegtgggtgg gtcaggagtg 31740 cagatctctg cagtgagtga gaagcctggg ataatgtgga ggactcctgt cccaagctgt 31800 tccctcctgc cccttccaca gcagctgcct gggttggctc atgtaaggga gctgccctgc 31860 tacctcggcg ggctcctggc cctgctccca ccgcctggct ttctgggtcc ctgccaggtc 31920 tccagcccac tggccttgac tccagagcat tccccctcac cccaggccct tatctggcca 31980 cctacccctc tcttcecctect cccttgette tectettggtt cccacctgag cccctggcat 32040 ctgccctgct cctgtgcccg ctgcatgtgc ccgtgagttg cggtgggtct gtgcatgtgc 32100 gcctgtatgt gtcggcaggc gtgtgtgttc ctgccagtca ctagcagtct ctccatctct 32160 gggtgtctct gtctgactct ggctggctca gcactggacc ggctgggcgg gggtgtcagg 32220 gagaccatta atctgcggtg acaggcccgg ctgetggegg agggggaggg gctcaggctg 32280 Cgggagcgcc gctgcaggag cctgggeggg CLgCggggga gggggccgct ggctgggcac 32340 CggCggSECg ggggagccta gtgtggeggg gLgggggtgg ggacctggta gaaggaggeg 32400 ccggctggag gagctgaggt tccgagtgcg gccgctgctg ggetggeggg Cgggcagagc 32460 acggcaccct ggcagcaggg cccacgccac ggggccatgg gcagctcgag CCaggcaggc 32520 tgctgcccac gcttactgcc agggtgaccc cagccctggg gcccagccac aaccaccctg 32580 gcttcatgcc aggggctgct ctggttgcca gtcggccagc ctcgggggtg cagcctgggc 32640 tgggactgct gctggggtgc aggtgaggca gLggCCgggc CCtCaggccC Cagggcaggc 32700 Pagina 158

P318031NL_ST25{9969840} (1) aggctgcagg gagccaagtc ctccatggcg gccccagecg ggaaggcgag caggacaggs 32760 gctctgcggc ccagggccca gaaaggccgg gtgaggcggg ccgtgcgcat ctccagcctc 32820 gtggcccagg aggtaggtga cacccggtcc ttcctcttct ttctggggcc ggcacactga 32880 ggcaggtggc tggcccaagg gctctgactc caggctgggg agagggctgc tgggaagtag 32940 gaagctttgg gagccgtggg gcctggccct gctcacccgc gcctggctcc tgttctcatt 33000 ccctcccagg ctgggcttac agagtttccg ttgctgggac ttccccgctc ctgtgcctgc 33060 catggcccac agtcctggag ctcaaaatgc aaagtgggga gatgacagga atgagatggg 33120 tgcccagaga gatggtggegg aggcggagtg ggacaaagga gcccatgggc aggaccagga 33180 gaagccggca ggcgacagct gggcctggca tggaggcgcc ggggctcctt tgctctgtgt 33240 gctggggttg gaccctgtgg gcaaaggagg cttgtgctgt tttggggggt cgaggaggct 33300 ggaatgtgcg gaagccactg tgtgggtggt gagggcgcgc tgggctgagg cacctggctc 33360 atctcttcat gggcagccga ggtgtggtgg ctgggccatg cactactctg tgacctcctg 33420 ggagccatcc agccgctctt ctgtcagcct ggggcaggct tgggccgaca gtggaggagg 33480 agtgggaggt ggctcagggc taagtctggg aatcccatcc ctgcccccac caccccacat 33540 ctgcacattc tgccttggct catacactct ttccagctgg gggacaggta caggtaggtc 33600 ttcccaccca cagctgagga gtcagggttt ggcttaagca agccagcttc agtattctat 33660 aggacaacag gctgggggga cagacctggc ccaaggccat aggtgagctg gtggctgcag 33720 agctgggact ggcccctgtc aggtgtgccc catgcacgtg tgagtttgta ccctggggca 33780 gtgttgctca tgctgtgtgc agactcaggg gtagggtcac tgctgttagg ggtggtcctg 33840 acacctccgc ccagaggctt gacctctcag gcacagagaa gagccctgtc cCaggggcagc 33900 taccttcttc tgccccctta tcagcagggg cctggccagt gttttctttt ttttttttca 33960 gtccccactt gatcactgaa gggccaccca cccatgggca tttgctctct gccttaagga 34020 gctgggcact tagacagcag acagagcaga aatggagcct gtgagcctgt ccatgtgttc 34080 cctccctgcc ctaggcctca gctgctcccg cagcccatct ggtaccgctg gaggccctgt 34140 gtccacccca gggtggtcaa atctttgact atgacccgtc ttgacttcta gttatttcct 34200 ctaaggagat gttctcccag cagcgccatc tgtgaaggtg tcttgggcaa gcccagcttg 34260 Pagina 159

P318031NL_ST25{9969840} (1) agtatgaagg ggagtctttg ggactcatca actatggcct ccccaaccca aaagcctgag 34320 getgtggggg cctgggagtc agtgcttctg ggagcagggc cagcacctct ggaattcatg 34380 geggctgggc aaggccttgg gttcagctgg ctgcccaggg atggcaggga gagcttgggg 34440 agggggcttt tgggttgagc tgcatgcctg agagaggatt ctagggagac ccagaagctt 34500 ctgggacagt gtggagaagc tggccggagc tcttcttagt ttgccttggg agcaagaggg 34560 acgtgcctgc tgtcaccggg tccggatggc cagcctgcct ctgtctgatg actgtggagg 34620 aggcagagtg tgagtgtgag ctggtacagg ggctgtgtgt gtcactgctg agcagtccct 34680 gtgtgcacgt ccctctgtat aagttgggtg ccatctcctc ccagctgggc tatgtcttct 34740 ttgtcccttt gtaagagggt gggaggatga ggactgtggg ctgtcccgga cctgtgtgga 34800 gagggggctg agggagcgag gtgtgcgagt ggagggtggg agtgtcttgg tctttctcca 34860 tcctggttgc ctggtgtcct cgggcctgtg gcatgaagag gggctggtaa gaactgcagt 34920 gactaccagt cagtggattg gggctatggc tcctggggcc actgcccagg ttgggagctg 34980 ttgtccttgg aaccctttcc ggtctgagac tatggcgtgt gtggttgggt gtgtgggcca 35040 gcgtctcagg gcagtcacag tggcaggtct gcatctgcat tgcecggeggg gagcagcact 35100 gegtgttcct gccgagtggc ctgaatccac gtctgccggg aaatgttgga actgtttgga 35160 gccagtcctg tgctgtgtgg cattttggga accatgatgc ccaaacctgg gggtggccat 35220 ttgctgtggt cgcttggctg aggggtgggc attctgatgg aagctttttt tggcctcctc 35280 ctcattctgc ttggccccgg cagagatgtc atcgctcctg cccccgaggt cccatggcct 35340 gcctcaccca cacctccgcc ttccccgggt gcaggtcctg tccctgggcg ccgacgtgct 35400 gcctgagtac aagctgcagg caccgcgcat ccaccgctgg accatcctgc attacagccc 35460 cttcaaggcc gtgtgggact ggctcatcct gctgctggtc atctacacgg ctgtcttcac 35520 accctactcg gctgccttcc tgctgaagga gacggaagaa ggcccgcctg ctaccgagtg 35580 tggctacgcc tgccagccgc tggctgtggt ggacctcatc gtggacatca tgttcattgt 35640 ggacatcctc atcaacttcc gcaccaccta cgtcaatgcc aacgaggagg tggtcagcca 35700 ccccggccgc atcgccgtcc actacttcaa gggctggttc ctcatcgaca tggtggccgc 35760 catccccttc gacctgctca tcttcggctc tggctctgag gaggtggggt cagcaaggag 35820 Pagina 160

P318031NL_ST25{9969840} (1) gcaggtggtg tagggagagg agaggccagg aatgtggtgg gaggtagtgg ggtggagsss 35880 gtggcgagga ggacagagaa aaagagagag agagagaaag agagaaagac agtgggagag 35940 agacatggtg gcaaggcagc tgcagaggca aggtgggtac cccagcaaac ctatgctcct 36000 tctctccaca caagctcctc gcaggttgca gagggccaca caggacacct ttttttctaa 36060 ttegtccatc ccaaggggcc attattttgt agcgtcacca aagtgcccta gaagctggtg 36120 ggagtgggca ggcaccctag ggtgatcact gcacctgtca gtgcccctgt ttcctcatcc 36180 acactatggg ggctgcctct gtcccaaagc tagcactaag attaatgaga cacctgtgct 36240 gggcccagtg tgggcttcac ctcttaggag gagggtctag gaagtctttg gggatctgac 36300 ctctgatgct cgctctgagg gccgcagcac ctcccctcct cccttgcccc ccttgcccca 36360 tcaacggaat gtgccccttc cctgtccccc agctgatcgg gctgctgaag actgcgcggc 36420 tgctgcggct ggtgcgcgtg gcgcggaagc tggatcgcta ctcagagtac ggCgcggccg 36480 tgctgttctt gctcatgtgc acctttgcgc tcatcgcgca ctggctagcc tgcatctggt 36540 acgccatcgg caacatggag cagccacaca tggactcacg catcggctgg ctgcacaacc 36600 tgggcgacca gataggcaaa ccctacaaca gcagcggcct gggcggcccc tccatcaagg 36660 acaagtatgt gacggcgctc tacttcacct tcagcagcct caccagtgtg ggcttcggca 36720 acgtctctcc caacaccaac tcagagaaga tcttctccat ctgcgtcatg ctcattggct 36780 gtgagtgtgc CCagggEgCgg BCggCgggga gagcccacgg tggaggaacc caagttggag 36840 gaaactgagg ctgctagccg ggccagagtg aggccctgga acttagagac tccaggggcc 36900 accatcgtga catggtttgg gggctgggtc accagggcgg ggactactga agggtggggt 36960 gtgctggagc gcagatgtac aagggcgtct cgtgggcatg tcgacgctga gactgagaca 37020 ctgacctggt gcggggcctg agcagggtcc ctgtgggtgg gtggggtccc tgcagaggct 37080 gacggcccca cgtgcccacg cccccagccc tcatgtacgc tagcatcttc ggcaacgtgt 37140 cggccatcat ccagcggctg tactcgggca cagcccgcta ccacacacag atgctgcggg 37200 tgcgggagtt catccgcttc caccagatcc ccaatcccct gcgccagcgc ctcgaggagt 37260 acttccagca cgcctggtcc tacaccaacg gcatcgacat gaacgcggtg aggccaccag 37320 agcgtggcca gtgggtggca ggctgggaag agtggggtgg cagaggggaa cctcttggca 37380 Pagina 161

P318031NL_ST25{9969840} (1) cagcaaacaa gtctcaggcg gtcacagtca gtagtaagga ccctggaggc acgttgcccc 37440 tttttgccca tgaggaaatg gaagcccttg gaaccccttc cctggagagc tccccttttg 37500 gegtgggagg tatatgtcac ataggcgatg gggttaaatc cttgtgacag cccagggegg 37560 gaatggtctc tatgccttac aaataagaac atggaggtct agagcccaag ggtgctcggc 37620 caggcatggt gaagctggag tggggtccca ggtccttccc aagacactgg gccccctgat 37680 gcttccgaga tctcccaggc ctggaggttg agatttctct gacatggagg ggtgggatgg 37740 tggagtagag tgtgggttgg ggggtcccaa gggagggtgt gctgagctgc ccccaccctg 37800 cccccaggtg ctgaagggct tccctgagtg cctgcaggct gacatctgcc tgcacctgaa 37860 ccgctcactg ctgcagcact gcaaaccctt ccgaggggcc accaagggct gccttcgggc 37920 cctggccatg aagttcaaga ccacacatgc accgccaggg gacacactgg tgcatgctgg 37980 ggacctgctc accgccctgt acttcatctc ccggggctcc atcgagatcc tgcggggcga 38040 cgtcgtcgtg gccatcctgg gtatggggtg gegggcgggc actggactgg aaatgccctc 38100 tgcagcctca agaggtgcga gccttctgaa tatgcagtca ctggggctgt ggacctggga 38160 ctgcctgcag ggtcactggg ctcctttaat tcacctaaac tcaggccctc caagcggggc 38220 catggagagg agccccacgt ggggtgaggc tgctgaactc tggggttccc acattctcct 38280 tcccttcagg atccgccaca aacagacact ttttgcttcc ttaaagtagg atcaaatcta 38340 gatcctctag cctgggcagt agaggaagaa atgctagcct ggaagctcgg catttggttt 38400 cactaagggc catgtggttc cctgcagcct catgcctggc cccttgacac atccaaagca 38460 aagggagtcc tgccccctcc ccccacttcc tttctaccct gcctgtgcac agtgggtggg 38520 ttggtgtgtc tggacactga ggacttcctc cccctttgcc tgtccttccc tcggccctgt 38580 gtgcctcagg gcagatatag caagctcttt cgaccatagt tgatggtagg acattttaga 38640 ctttgtttct cagctctgta caaacacaaa tacacacccc cacaaaacta aaatcaaagt 38700 ttcactacat aacactgggc cttactgcat gtggttcatt ctagcatttc tgttctgtgc 38760 tgtgctaagc tatactactg tatgttcttt cagtaaaaaa aaaaaaaaaa aaaaaaaaat 38820 gctggttttg attcactact gtgttctgat ctttggtttg aagaacattg cttataaggg 38880 tgcagtgatt ggctaagagg gtgtttggga cctggggttt ggggtagtcc tgctggctgg 38940 Pagina 162

P318031NL_ST25{9969840} (1) gtctcatttg ctgggagacc ctgggctcct ggcccctctg gcactgtcgt gcctgctggg 39000 agctgaggga gggtgggetg gcagattgct tccccggttg tgtgggttgg gggccggctg 39060 gcaggcgacc cagctgaggg gacatgctct gcccggggtg gggatgecgg gagaaggtgc 39120 ctgctgcctg gatgccgccc cccggggctg agctccctgt cctctccatg gcctccaggg 39180 aagaatgaca tctttgggga gcctctgaac ctgtatgcaa ggcctggcaa gtcgaacggg 39240 gatgtgcggg ccctcaccta ctgtgaccta cacaagatcc atcgggacga cctgctggag 39300 gtgctggaca tgtaccctga gttctccgac cacttctggt ccagcctgga gatcaccttc 39360 aacctgcgag atgtgagttg gctgccctga gctggacccc atcctcctgc ttccagctgt 39420 gtggctgccc ctttgctgtg tgacattgaa tgtgatgctt ctctgtctca gtttccctgt 39480 ctgtcaaatg gtatcataga gcagcctaca aaagtcccaa actcagtctt tgcctggggc 39540 aaaatcacat tgggggcagg gaagggtttt cccagagctg gggcgcccag ccctactttt 39600 tctgtgttaa ggagggagct tggggcctga ccccggLggg gcaggagagc actgaaaggg 39660 ccctgatact gattttggtt ccagaccaac atgatcccgg gctcccccgg cagtacggag 39720 ttagagggtg gcttcagtcg gcaacgcaag cgcaagttgt ccttccgcag gCgcacggac 39780 aagggtgagg cgggggageg gaggaaggeg BaggSCgSEEg acaaggtgag gctgggagct 39840 ggaaggcggg gtgggccgtg tctgctttac ccaggcctca gaccttccca tcccttccte 39900 cagtgtgccc actccacacc ccagctgtcc agatgctgcc catctttcag ggcccctgca 39960 gctgtcacct cctccaggaa gccctcttcc accctccagg gcaagcctag gctttttgga 40020 cttcacaggg ctggggtctc tgtgagcact gaggaggaaa gaacatagaa caaaggaggg 40080 ccagggagcc aacgatgggg ctgaggcccc gcagtcccag agatccgcca acccctgaag 40140 caccattgcc agtgcttggg cagtcagggg gtcctgggag ttcttggtcg gcttgacagt 40200 gggtgcaaga tagcagaaga agcgactggg gacaaaccag ggcttcctgc ccagtcctct 40260 ctcgggctcg ctcctgtccc ctcctccacc ctcgccccct cctctctgtt ctcctccect 40320 ctctgaggcc cattctctgt ttcccacaga cacggagcag ccaggggagg tgtcggcctt 40380 ggggccggge cgggrgggeg cagggccgag tagccgggge CggCCggSES ggccgtggss 40440 ggagagcccg tccagtggcc cctccagccc tgagagcagt gaggatgagg gcccaggccg 40500 Pagina 163

P318031NL_ST25{9969840} (1) cagctccagc cccctccgcc tggtgccctt ctccagececc aggecccceccg gagagccgcc 40560 gggtggggag cccctgatgg aggactgcga gaagagcagc gacacttgca accccctgtc 40620 aggtatcccg ggcgacggge gggcgaggga ggaccgggcg tggcagcggt ggtgcgtcta 40680 ccccgctcac ccagctctgc tctctggctg caggcgcctt ctcaggagtg tccaacattt 40740 tcagcttctg gggggacagt cggggccgcc agtaccagga gctccctcga tgccccgccc 40800 ccacccccag cctcctcaac atccccctct ccagcccggg LCggCggCCcC cggggegacg 40860 tggagagcag gctggatgcc ctccagcgcc agctcaacag gtgagggagt gcaggtggges 40920 tgggggggca cgccctggag tctggtccag gtcctggcgg tgttgtctgg tagagggaga 40980 gggcctgtct gtgtgtgcag cagctggatc cccttcttcc attcctagcc cagctggggg 41040 ctgccacacc tctagcgcgg agggcctggg ttgacaggct gcccctcccc cctccccttc 41100 cccttcccca ctcccggtgg aggctgtcac tggtgtcccc acttctctga gcatccccca 41160 cttcctgccc caggctggag acccggctga gtgcagacat ggccactgtc ctgcagctgc 41220 tacagaggca gatgacgctg gtcccgcccg cctacagtgc tgtgaccacc ccggggcctg 41280 gccccacttc cacatccccg ctgttgcceg tcagccccct ccccaccctc accttggact 41340 cgctttctca ggtaagctcc agccctgctt cctcgatgta ccgtgacccg cttgttcccc 41400 gattccagcc caaacctgct ttgctgcctt tctgctgggc tctgtggccc agcctccctg 41460 aaccttgttc ctgccctcct cccacccctg tcctcctgga ggaaggctgc ttcaggagct 41520 gcaggctcag ggtagggcag gagagtcccc cgtccaaagc ccctagacag ctgggtgcag 41580 ggaacctgta ggctaagtgg cccgcagctt cactccccta gtgtccacag cctccaccgc 41640 caggccccgg agcatcgcct gaagagccag ctccaacggt ggtgcctgtc tcacggccac 41700 gegtggtctc caagaactga ctgagcccga gacgtggcgg tggaggctgc ataccagagt 41760 atctagggac agggtcccgg gtcccttctg tgagtgacac tccctggtct caccgtcctg 41820 cccatccttg cccttggcag tggctgtcac agggtcatct tgagggtgct tggcatttcc 41880 cagggtgcag aggccaagcc tccaggggag gacttcctgg gcctgctttg ccccctctgc 41940 tagagatggg ccttagttgc cccttgtcct ctcctgggtg gacctgcatg ttcctggcct 42000 ctgcaggatg gcggtgtctg tgtcaacctc cgcgtccatg gtggggactc ctctggctgc 42060 Pagina 164

P318031NL_ST25{9969840} (1) cagaggcggc ctgggatctg ctccctcccc acttcccttg gcatccgctc ccccctccca 42120 tatgctgcct gctcctgttt gcagacacga gggcccacgt cagcaaatgt gccagtctcc 42180 cttcgcagcc gcccccgcag agctctcacc agcagacgct cagtacccac aaacccctcc 42240 cgcctgcccc ggccctctcc ctggccttca gatgcctggc cgccagcctg cctctgtttc 42300 taggtccccg agttctttct ggattgcggg aaggaatctt gtctcttttt ctgtcccagc 42360 ttctgtccca acacagagac tgcttgttcc aggttgggtt gggctcagcg gccacattcc 42420 ctcgggaaga gacaggggcc tagggttttc cctgctccca gctctcttgt ttggctcctg 42480 ctctcagaga atgcctttct ctacttccca ccttggtgcc tccccttggt ggcccctgtc 42540 ctgctctcgc tgccttggct cttgtcccct ccagcttctc tgcccacctg actctcctct 42600 tggccctcct gtcctccegt ccatcctctg tccccgggtt ctcctgcccc tttccctccc 42660 cttcctcctec ctccatggcc tcttcgcctg cccatgctct gtgtgtattg caggtttccc 42720 agttcatggc gtgtgaggag ctgcccccgg gggccccaga gcttccccaa gaaggcccca 42780 cacgacgcct ctccctaccg ggccagctgg gggccctcac ctcccagccc ctgcacagac 42840 acggctcgga cccgggcagt tagtggggct gcccagtgtg gacacgtggc tcacccaggg 42900 atcaaggcgc tgctgggccg ctccccttgg aggccctgct caggaggccc tgaccgtgga 42960 aggggagagg aactcgaaag cacagctcct cccccagccc ttgggaccat cttctcctgc 43020 agtcccctgg gccccagtga gaggggcagg ggcagggccg gcagtaggtg gggcctgtgg 43080 tccccccact gccctgaggg cattagctgg tctaactgcc cggaggcacc cggccctggg 43140 ccttaggcac ctcaaggact tttctgctat ttactgctct tattgttaag gataataatt 43200 aaggatcata tgaataatta atgaagatgc tgatgactat gaataataaa taattatcct 43260 gaggagaact ccagtggtgc tgggagggtg taactgttac ttgctgtgtc ctgtgttcct 43320 gtctggacat gcataggtgt gcctgcacag gaatccgggt ctgtctgcat gcccaggccc 43380 acagggctgc ccctgtatcc aggtctgggt gtgtgcagtg ggggtgctcc ctgggggetg 43440 acacaggtca tctccaggga gacagaatct tgtctctgta gggccctgga ttcaagataa 43500 caaaaaaccc cctgaaccac atgcatttag aggcagtcct gatgggacac tgacctggtg 43560 tctagcccct gagggccatg tgcctctgct gggtggggca ccctggctcc cacagcttgg 43620 Pagina 165

P318031NL_ST25{9969840} (1) cttcacactg actgtggaag cagccctgca cttgtctccc tcacccagtt cctgagcctg 43680 cgttgaccct aacctgaaat tggtctgagt cctggctccc cctgccaccc acaacttagc 43740 cttatcttat ccaatacaat tgggccccgg acacactggt gactccagcc ctgaccagag 43800 ctcacctggt gtctggctgc agatggacct cacttgtatc accttggtcc cctgtgatcc 43860 tggatctcaa gaggagcttc ctgaagaggg tgcaaccttc agggtgactc attcccatga 43920 CCggggagca aggcaaggca gatgtcttgg ggacagtgaa gggtgtgtgt gcaagtgcct 43980 gcgtgtgcct gtgtgtgtgt acatgtgtgt gcatgtgctg ctgggcttgt aaggacatgc 44040 agcccccaga ggaatgtggt gcaatgactg aggctggtgt ggagagccaa tgggtggcgg 44100 gcaggctgtg cccagctggg tgggtttgca ggcagggagc tgccaaaggc ctccagagac 44160 agagcagctt ctcactgagt ctttgaaacc tctgaccctc ggaggtgggt ttgttgactg 44220 ttttatttta ttaatttttt tttgagatgg agtcccgctc tgtcacccag gctagagtgc 44280 agtggcacaa tctcaactcc ttgcaagctc cgcctcctgg gttcaggcca ttctcctgcc 44340 tcagcctccc gagtagctgg gactacaggc acccaccgcc acgctcagct aattttttgt 44400 atttttagta gagacagggt ttcactgtgt tagccaggat ggtcttgatc tcctgacctc 44460 atgatccgcc cgcctcagcc tcccaaagta ctgggattac aggtgtgagc caccgcgcct 44520 ggctgttgac tgttttaaag acaactgtgc cgttctcact gcccatgcct gggtggggeg 44580 ggtgttggtc tctgagcagc ctggaaggtt ccctgtgccc agagcaggga ggagagaagg 44640 aagggatctc cccctttcca gggcctggga gagacaggga gaaggaagag atccacaggt 44700 Cagaggggga gaggggtgac agagagggat gccaggcaga aaagacaggt ttgtgcatca 44760 ggatctgtcc cacacatgta cacacatgca catgtgagtg tgagcttgct tctgctcatg 44820 gtttaaaagt ttaacacagg ccttcctggt tggttgatgt gactgtcact gcaggtgggg 44880 gcctagctgg ggtggcctag atttggtgtc agtggggcat ggctctgctt atggactctg 44940 gagcccaggg caggagacag acccaggcag tggcttatgg ggagcagtga aggtacacac 45000 gaaactcaat gctcttcgca tgctgggctg tgagctgagc caactccccg ctccctggtg 45060 ctgtttcatc cttgtcatgg tctgcccttg tccttagagt gccttcatct tgggagtctg 45120 cctgatgccc accactccgt gtccagcagc cgggctgggt gttggtggac acagagagga 45180 Pagina 166

P318031NL_ST25{9969840} (1) gaggaagaaa gggcctttgc ccccctcctg gctttgtagc agaggtgcca ccggttattt 45240 ctttgtggca ccatgccagc accacggtta gctatcttcc cctaggtgag ggccctgcct 45300 gegcggtgga ggtctggcgt ggattttggt gctcctgagg agtggatgca tgtgtgtgag 45360 tgagtgtgca tgtgtctgtg ctcaggatgg ggtctgggcc cagcagaagg caacctgtcc 45420 atgctggtcc tcgatagaaa tacacaggtg tcccggagtc agttcagtct ggatgggtca 45480 ggctgcccag atgccccttg ccaagtccct tccaggactc ccctgccaag gctggagagt 45540 gegtgagggg cagtcacagt ggctgcgttt gccaggtggt ccctgggtcc ggtatgcagc 45600 tgggtgctct gcccttgtgt cctgaatccc taccccagcg gagggcatgt tagcatccac 45660 atgaggacag tcagtttctg gctggtccag tgactggctg gaggtcacat ccctagttgg 45720 tggcaggtgc gtcagacttc aggttctccc cacccatcct cccgcctgca ctgcccagcc 45780 acaggcaggc ctgctccttg gctcctcccc tcatctgggc tcccagagcg acaggaagtt 45840 tggtggtgag gacaagggca agccgtggtg gtgcagtccc tcccctcccg cagggctggg 45900 cacctggctg ttaagggcct gtcatccaga gctgagggtt ttccccaacc aagagagcca 45960 gagggtggga cgggagctgg agggagctgt ggcaagagag gaagggatcc atgtggttgt 46020 caacacgtgg cagaagcctg gcgcagggcc ccaagctgga gtctggtcag gggetgggag 46080 caggaacaga agcaggaggg cagcgtggcc tgtctcgcct ccacctccat ccccaggcgt 46140 ggagggatgc ccagcctgcc tatccacgtg caagctggtc tccagcacct gtccttacct 46200 aagatctcat ccccactcag ccgggtggtg gcagctctca catttcactg acaaataaca 46260 gcaatggata gttcctgagc acgccctttg cggcaagcat tattccagcc acctgaaacc 46320 aaagccccag aggctgagtg acttgcccga gcatacccca ctcctgagag tggagctgga 46380 aggtgagccc aggtggtctg agtcacatgc ctgctggtcc tgaagggaca ggtggacttc 46440 tcccccttgc ctgcagggca aggccctctg tgccagtttt gcacagaggt tctcatcttt 46500 ctttccagac tcacctgccc tctcccctcc ctgccatgtg ggtcatgctg cagccacgcg 46560 agcttccgtc ctgttcacca tgttcttgca tctgccctgt cttgctctct tcttgtgacg 46620 cagactccct catccttggt gtggaaagac ccttctgtct tcccagccct agcatggacg 46680 tcgccgctcc tgaggccttc tgcaggtttc tcagccggtt gtggcactcg ctggcacctg 46740 Pagina 167

P318031NL_ST25{9969840} (1) tgttaggggc tccccacacc ctgcctgctt gctcgtctgt cctccgtaaa ggggaagggc 46800 tgaatgctgg tcatcttttc gcctattccc acccagcatc tagcacagtg cccaggacct 46860 gatgtggcca ctaggcagcc cacaagaccc catgtttgag gattttgcac agaaaaggga 46920 gatgtcttcc tgacagagtt ggctgggcta aaatttgcgt ctgtccctgg aggtcacttt 46980 cttcccaaag gcttggcctc tggattatgg ctgcctcccc gcctgcctcc ccgcctgcct 47040 ccccagggag cacaagcccc actccttcac tggcaatgtg gctacacatg cttatctttt 47100 tttttetttt tactttagca caaccaaggt aaatattaaa tatttgaaag agaataacgg 47160 gagcagtggg gaggataaac cagagacttg gaccgagagc ctcaagtcca ttcattttgt 47220 agattgaatg ttggggcctg tccatggctg ccccagtctg cgggcttgtg ggccccaccc 47280 tgttcccatg ctctggaatg cctctgaggt ggaactgggg atactttttt gcaggggggt 47340 ttgctcccag tttccagttt ccaggtggcc tctgagtgga gggctccagc tcaccgtcct 47400 ctgggtggtt gttcatttgt ccttcgatgg tcaaggctga gggaggttac accgggggct 47460 ttgcagttcc agattcgggt ctgtgggatc taccttttcc cctggtggtg gacagtcttc 47520 cggaaactga aacctgattg aacttgcttt tcagtgagag cttacactgc tgtaggaaga 47580 ggtatttctt tgtaggagtc ctggcagctt gacaacacaa ggaccataag aggacacatg 47640 tggtgtgtta gtgtctctca agtgctcaac gatgttgaaa taaacggaaa acgcagggcc 47700 ttccaaagtg actgttttga ggcattttta gataattgta tatcattaaa gagtggttag 47760 tgtgaaaatt gatttagtaa aagattaaat agtaactatt ttcttaaagc tgcatatatg 47820 cctggtatta agcgataaaa actcaattta aggctggatg cggtggctca cgcctctaat 47880 cccagcactt ttgggggccc gaggcaggtg gattgcttga gattaggagt tggagaccaa 47940 cctgggcaat atggcaaaac cctgtctcta caaaaaaata caaaaattag ccagcatgat 48000 agcacgcacc tgtagtccca gctacttggg aggctaaagt gggaggacag cttgagcctg 48060 ggaggcggag gtttcagtga gctgagatcg caccactgca ctctagactg ggcaacagag 48120 ccagaccctg tctcaaaaaa acccaaccct cccaccccca aaactcaatt tatcttgatc 48180 tttttattac agtagcattt taaagagggt gcttcttaat cattttgaat tactaaacat 48240 ccaagtacaa ttctaggttt tttgttgttg ttgttgttaa cttttgtctt ccaaaaaaag 48300 Pagina 168

P318031NL_ST25{9969840} (1) aaaaagagaa tgcattctga ggcacttgtt taaataatca gcttcattat tttatattaa 48360 taatcacacc ttataagtga gttggaagtc tgcaagagac attgctaaga aaccagagca 48420 gaagaattta gcatgatcct cttaccatta ttttaaaatt agcaatagca taaggtctgg 48480 ctgcaaaaga cttagatgct aactggcaag aaaatcagtc attagggaaa cagtctggag 48540 agggctctca tagcaacaca accatatgtg acagctctgc ttcccctttc tgctctacca 48600 ggtgatggca gccagataat cagatggaac ttgctgatga gggacactag gaaggtcaaa 48660 tcagggtggt ctcctggact taagcagccc aggtggaatg gaagtcttag cagagaggct 48720 gtatttaatg tcaagttacc ctgggtgccc tgggatgatg actcttccgc tgagaagttt 48780 ggctttctgt ggggctctaa gtgaccttag ggaagtgtta ttctcaggct ggagaaagga 48840 gcatggagga atgaggatta ggcgacatgg gaatgagtga agggactcag ctgtcctcca 48900 tcctctggcc ctgggggagc tttttctttc agatactgag aaatgggttg agcccatgtg 48960 cctagtgcca ccccatgtct ctggggatgc ctgaggggca ggggcgaggc ttatgttctg 49020 agtcaaggca cctgagacat tggagtcagc aggcactggc tcctaagcat gcagctggtt 49080 cctgtgcctg ggaggattgc tttagctcag gggttcaaga ctagcctggg caacaggctc 49140 ttaaaaaaat aattaaaaaa atcagctggg cattgtggca tgtgcctgta gtcccagcta 49200 cttgggagac tgaggcagga ggattccttg agcccaggaa gttgaggctg cagtgagtca 49260 tgatcgcccc actgcactcc agcctgggca acagagcaaa ataccctgtc tccaaaaaga 49320 aaaaaatgtg aggggccttt gtgagcactg gggaagttac ctcatgatac aggccagaaa 49380 acaacttaaa agatgatccc agaccttcaa gccctaagtt cttggccacc ctgtcagtgc 49440 cacctcactg gtgacctttt tccttgtcct cccacagcca ctgggatgct atccagatgc 49500 atctgatgaa ccccagggag gaattaaaac atggccctgg aagggaggag aggctgcagt 49560 atggcttctg tcttggtcag caagcagttt tcccataata ataattagcc atcataatga 49620 ggcagcacgt tgcagagatg cggagctggc gaggatgagg aagctgcaaa ggaactgtgc 49680 tgaatgggag gcacaggatc tcctgggaga gcagagcctg gtgcaggtct gagggttggg 49740 ctgggcttgg ctcagtggct cctgtcatgc cttaaaggca gtcctgaggc gcacaggaag 49800 tggtgcgtga tcgtaggctg agctgttcag ctcacggctt gcaaagccca ggtcttagga 49860 Pagina 169

P318031NL_ST25{9969840} (1) atatcccttc ctgagccaat tagcttccta cttgccgagg cagctgctgc agctttagcc 49920 cagcctatcc tcttgcaacc tcaggcatca ggtgaaggat cagtgtttgg aggggacggc 49980 aagtgccatt gcttattaga gactcaagtc acaccagaca tgactgagga ggtgttgggs 50040 cacagcgttg cttgctgagg acaaactcct cgaagtttcc tgctaacttt tcaccaactg 50100 gtaaacgttg aatgttctgg gtaactgcca aatttgtggt tcagaaatgg aaaaagtgca 50160 gaaaagctga aagtgacttt agcaaagagg cagctccttt ggctgaagtg cttggcaggg 50220 tggacgtaac tgacgcagca ggacagacac gcctctctcc tcagcatccc ctgtgggtgg 50280 cgtctccttg gtcctgggtc ttgggttagc cccgcagctg gatccggtgc tggtgaggct 50340 cctgtccagg tggcctcact ctagcagagc agactgggcc ttgtgggtgc ttaccatgcc 50400 cgggatcccc cgtggagact ttatacccca acccagggca ggagagcttt cacttaaaat 50460 gttttccttg agccccacct agaatcacag aaagtcgcca aaagtcatga gagagcacag 50520 tctaccccgt cgtttttcag tggaggggaa tgaggcttaa agttgctcaa ggcaggagtt 50580 tccagagcag cacggcccct cacttcccgt cctgctgcag gctggatggg atgcaaccgc 50640 acccttcagt agctcataat ttagctggga ccatataaga gaaataaaag aaaatgacca 50700 tggccccact tttatgaagt gcctaacaga gcttctttta ttcctattca agtcctcctc 50760 attctccagg acctggctcc tggcagccct ctccataaag cctctccatg aacccttgtc 50820 agtctacagc tatcctctgt ggcattgcat gacacacaaa gccacaccgt cctgtaactt 50880 tttttttttt ttgagatgga gttgagtttt gcttttttcg cccaggctgg agtgcaatgg 50940 cacgatctca ctgcaacctc tgcctcccgg gttcaagcga ttctcctgcc tcagcttcct 51000 gagtagctgg gattacaggc atgcggtact atgcccagat aattttgtat ttttatagag 51060 aggtggggtt tcaccatatt ggccaggcta gtcacgaatt cctgaccttg ggtgatccac 51120 ccgcgttggc ctcccaaagt gctgggacta caggcgtgag ccactgtgcc tggtcctgta 51180 acagtttttt ttaattaatt attatttttt taagagacaa ggtctccctc tgttacccag 51240 gctggagtgc agtggtgcaa ctatggctca ctgcagtctc aacctcctga gctcaagtga 51300 tcctcccgcc ccagcttccc gagcagctga gaccacaggc gtgtgccacc acacctggat 51360 aatgttttct cactgaaagt ggaaacagcc atttctgtct tctatattag cagaaaaaat 51420 Pagina 170

P318031NL_ST25{9969840} (1) atttttaaaa acccgaagga ctgaactttc agttcttact gcaaagcccg Caggggcagg 51480 gttatctgga ctggagggat gtccactctt tgctcacagc ctgactttga tactgtgttc 51540 tctgtggtct aaggaactta ccaatggggc ttcagtagtc tcatatccta aacacgttat 51600 cgagttgttt tcattttcag aaatcattaa gggacacatt tgacccacat agagactggc 51660 agggtcccag ttgcctgcta agttctcaga cagggccagg ctgatgaaaa ttcaacttaa 51720 gtcattaatt gctgcataat agcaatggtg ccgttgtgaa tggaggtggg gtccttagaa 51780 actggctctg gaggatgggg cagggacttg gctcttggag atgcggccat gagagctcct 51840 gggaatgaca tggagggaat aacaggtgtc ttgacactag cacctggagg tgtgtgaggg 51900 gccaggatgg actgcggata gaccagtcgc tgcaagaact cttcctcacg ataacttcgt 51960 ataggagact ttatacgaag ttaagcgctc actggccgtc gttttacaac gtcgtgactg 52020 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 52080 gcgtaatagc gaagaggccc gcaccgatac gtcaggtggc acttttcggg gaaatgtgcg 52140 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 52200 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 52260 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 52320 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 52380 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 52440 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 52500 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 52560 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 52620 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 52680 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 52740 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac 52800 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 52860 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 52920 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 52980 Pagina 171

P318031NL_ST25{9969840} (1) actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 53040 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 53100 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 53160 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 53220 tgagttttcg ttccactgag cgtcagaccc cttaataaga tgatcttctt gagatcgttt 53280 tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc 53340 gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag gagcgcagtc 53400 accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga ctaactcctc 53460 taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc gggttggact 53520 caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt tcgtgcatac 53580 agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga atgagacaaa 53640 cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg agagcgcacg 53700 agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac 53760 tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc 53820 tttgccgcgg ccctctcact tccctgttaa gtatcttcct ggcatcttcc aggaaatctc 53880 cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag 53940 tgagcgagga agcggaatat atcctgtatc acatattctg ctgacgcacc ggtgcagcct 54000 tttttctcct gccacatgaa gcacttcact gacaccctca tcagtgccaa catagtaagc 54060 cagtatacac tccgctagcg ctgcatctca attagtcagc aaccatagtc ccgcccctaa 54120 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 54180 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt 54240 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctttgcaaa gatggata 54298 <210> 198 <211> 9454 <212> DNA <213> Artificial Sequence <220>

Pagina 172

P318031NL_ST25{9969840} (1)

<223> pEfBOS f1p0 2A creIRESPuro plasmid

<400> 198 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta 240 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 300 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 360 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 420 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc 480 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct 540 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 600 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggegeg 660 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 720 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 780 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc 840 cttgcgtgcc ttgaattact tccacgcccc tggctgcagt acgtgattct tgatcccgag 900 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct 960 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac 1020 cttegcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct 1080 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact 1140 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt 1200 tcggecgagge ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct 1260 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggeggea 1320 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct 1380 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca 14409 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg 1500

Pagina 173

P318031NL_ST25{9969840} (1) gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg 1560 geggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg 1620 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg 1680 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga 1740 ggaattctct agggaattcc accatggctc ctaagaagaa gaggaaggtg atgagccagt 1800 tcgacatcct gtgcaagacc ccccccaagg tgctggtgcg gcagttcgtg gagagattcg 1860 agaggcccag cggcgagaag atcgccagct gtgccgccga gctgacctac ctgtgctgga 1920 tgatcaccca caacggcacc gccatcaaga gggccacctt catgagctac aacaccatca 1980 tcagcaacag cctgagcttc gacatcgtga acaagagcct gcagttcaag tacaagaccc 2040 agaaggccac catcctggag gccagcctga agaagctgat ccccgcctgg gagttcacca 2100 tcatccctta caacggccag aagcaccaga gcgacatcac cgacatcgtg tccagcctgc 2160 agctgcagtt cgagagcagc gaggaggccg acaagggcaa cagccacagc aagaagatgc 2220 tgaaggccct gctgtccgag ggcgagagca tctgggagat caccgagaag atcctgaaca 2280 gcttcgagta caccagcagg ttcaccaaga ccaagaccct gtaccagttc ctgttcctgg 2340 ccacattcat caactgcggc aggttcagcg acatcaagaa cgtggacccc aagagcttca 2400 agctggtgca gaacaagtac ctgggcgtga tcattcagtg cctggtgacc gagaccaaga 2460 caagcgtgtc caggcacatc tactttttca gcgccagagg caggatcgac cccctggtgt 2520 acctggacga gttcctgagg aacagcgagc ccgtgctgaa gagagtgaac aggaccggca 2580 acagcagcag caacaagcag gagtaccagc tgctgaagga caacctggtg cgcagctaca 2640 acaaggccct gaagaagaac gccccctacc ccatcttcgc tatcaagaac ggccctaaga 2700 gccacatcgg caggcacctg atgaccagct ttctgagcat gaagggcctg accgagctga 2760 caaacgtggt gggcaactgg agcgacaaga gggcctccgc cgtggccagg accacctaca 2820 cccaccagat caccgccatc cccgaccact acttcgccct ggtgtccagg tactacgcct 2880 acgaccccat cagcaaggag atgatcgccc tgaaggacga gaccaacccc atcgaggagt 2940 ggcagcacat cgagcagctg aagggcagcg ccgagggcag catcagatac cccgcctgga 3000 acggcatcat cagccaggag gtgctggact acctgagcag ctacatcaac aggcggatcg 3060 Pagina 174

P318031NL_ST25{9969840} (1) gcagcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag aatcccggcc 3120 ctaagaagaa gaggaaggtg tccaatttac tgaccgtaca ccaaaatttg cctgcattac 3180 cggtcgatgc aacgagtgat gaggttcgca agaacctgat ggacatgttc agggatcgcc 3240 aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt ttgccggtcg tgggcggcat 3300 ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc tgaagatgtt cgcgattatc 3360 ttctatatct tcaggcgcgc ggtctggcag taaaaactat ccagcaacat ttgggccagc 3420 taaacatgct tcatcgtcgg tccgggctgc cacgaccaag tgacagcaat gctgtttcac 3480 tggttatgcg gcggatccga aaagaaaacg ttgatgccgg tgaacgtgca aaacaggctc 3540 tagcgttcga acgcactgat ttcgaccagg ttcgttcact catggaaaat agcgatcgct 3600 gccaggatat acgtaatctg gcatttctgg ggattgctta taacaccctg ttacgtatag 3660 ccgaaattgc caggatcagg gttaaagata tctcacgtac tgacggtggg agaatgttaa 3720 tccatattgg cagaacgaaa acgctggtta gcaccgcagg tgtagagaag gcacttagcc 3780 tgggggtaac taaactggtc gagcgatgga tttccgtctc tggtgtagct gatgatccga 3840 ataactacct gttttgccgg gtcagaaaaa atggtgttgc cgcgccatct gccaccagcc 3900 agctatcaac tcgcgccctg gaagggattt ttgaagcaac tcatcgattg atttacggcg 3960 ctaaggatga ctctggtcag agatacctgg cctggtctgg acacagtgcc cgtgtcggag 4020 ccgcgcgaga tatggcccgc gctggagttt caataccgga gatcatgcaa gctggtggct 4080 ggaccaatgt aaatattgtc atgaactata tccgtaacct ggatagtgaa acaggggcaa 4140 tggtgcgcct gctggaagat ggcgattagc cattaacgcg gatccctcga ccccgggatc 4200 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 4260 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 4320 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 4380 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 4440 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 4500 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 4560 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 4620 Pagina 175

P318031NL_ST25{9969840} (1) aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 4680 tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 4740 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca tggcgcgcct 4800 taccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg 4860 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg 4920 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat 4980 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca Cgccggagag 5040 cgtcgaagcg ggggeggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc 5100 ccggetggece gcgcagcaac agatggaagg cctcctggcg CCgCaCCggC ccaaggagec 5160 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag 5220 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga 5280 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga 5340 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg 5400 CCCgCCCCaC gacccgeage gcccgaccga aaggagcgca cgaccccatg catcgtagag 5460 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 5520 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 5580 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gLgggEgcagg 5640 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 5700 tggcttctga ggcggaaaga accagctggg gctcgatggc gcgttctaga gtgagggtcc 5760 ccacctggga cccttgagag tatcaggtct cccacgtggg agacaagaaa tccctgttta 5820 atatttaaac agcagtgttc cccatctggg tccttgcacc cctcactctg gcctcagccg 5880 actgcacagc ggcccctgca tccccttggc tgtgaggccc ctggacaagc agaggtggcc 5940 agagctggga ggcatggccc tggggtccca cgaatttgct ggggaatctc gtttttcttce 6000 ttaagacttt tgggacatgg tttgactccc gaacatcacc gacgtgtctc ctgtttttct 6060 gegtggcctc gggacacctg ccctgccccc acgagggtca ggactgtgac tctttttagg 6120 gccaggcagg tgcctggaca tttgccttgc tggatgggga ctggggatgt gggagggagc 6180 Pagina 176

P318031NL_ST25{9969840} (1) agacaggagg aatcatgtca ggcctgtgtg tgaaaggaag ctccactgtc accctccacc 6240 tcttcacccc ccactcacca gtgtcccctc cactgtcaca ttgtaactga acttcaggat 6300 aataaagtgt ttgcctccag tcacgtcctt cctccttctt gagtccagct ggtgcctggc 6360 caggggctgg ggaggtggct gaagggtggg agaggccaga gggaggtcgg ggaggaggtc 6420 tggggaggag gtccagggag gaggaggaaa gttctcaagt tcgtctgaca ttcattccgt 6480 tagcacatat ttatctgagc acctactctg tgcagacgct gggctaagtg ctggggacac 6540 agcagggaac aaggcagaca tggaatctgc actcgagaat tcactggccg tcgttttaca 6600 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 6660 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 6720 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 6780 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 6840 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 6900 cctttegctt tcttccctte ctttctegcc acgttcgccg gctttccccg tcaagctcta 6960 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 7020 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 7080 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 7140 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 7200 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 7260 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 7320 cgacacccge caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 7380 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 7440 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 7500 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 7560 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 7620 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 7680 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 7740 Pagina 177

P318031NL_ST25{9969840} (1) aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 7800 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 7860 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 7920 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 7980 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 8040 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 8100 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 8160 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 8220 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 8280 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 8340 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 8400 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 8460 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 8520 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 8580 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 8640 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 8700 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 8760 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 8820 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 8880 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 8940 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gCggtcgggc 9000 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 9060 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 9120 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 9180 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 9240 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 9300 Pagina 178

P318031NL_ST25{9969840} (1) ttectggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 9360 gtggataacc gtattaccgc ctttgagtga gctgataccg cCtCgccgcag ccgaacgacc 9420 gagcgcagcg agtcagtgag cCgaggaagcg gaag 9454 <210> 199 <211> 1004 <212> DNA <213> Artificial Sequence <220> <223> Sequence of the genomic region surrounding the deletion of KCNH2 <400> 199 ggctttccca tccgtgggaa cttgaaattt cctccttttt tttttttttt tttttgagat 60 ggagtctcgc tctgttgccc aggctggagt gcagtggcgt gatctcagct cgctgcaagc 120 tcegcctccc gggttcacgt cattctcctg cctcagcctc ccgagtagct gggactacag 180 gctcccgcca tcaagcccag ctaatttttt tgtatttgta gtagagatgg ggtttcaccg 240 tgttagccag gatggtctcg atttcctgac ctcatgatcc gcccgcctcg gcctcccaaa 300 gtgctgggat tacaggcgta agccacagcg cctggccgaa attgtccttt attccgggga 360 tccagagtga gaacgagggg gtttcagggg caggctgagg cctcctgaat gctcaccata 420 ggtgccctgt cttgggctct gaaggcccta catacaaagt catcctatct gctgaggcaa 480 agatataagg ttacccagca cagtcagcac aaccagatgg ccatgtgctc cggggccaga 540 gggagacgga gggacactgt ggcccagatg taaatgatgg atttgctctt tttgtatttt 600 attttatttt ttgagatgga gtctcacact gttgcccagg ctggagtgca gtggggcgat 660 gtcagctgaa gcgattctct gcctcagccc tcccgagtag ctgggattac aggcgtgaac 720 caccatgcct ggctaatttt tgtattttta gtagaaatgg catttcatca tgttggccag 780 gctgctcttg aactcctgac ctcaagcaat cctcctggct ctgactccca aagtgctggg 840 attacaggtg tgggccacca tgcccggcca catctttttg tattttaaag tgaaatttaa 900 aaacacaggt taagggttaa aattatgaaa cagacctgat ataaaacatg ttattttggg 960 gataaatttt cctggttgct gttgtggaga aggtggaata tttc 1004

Pagina 179

P318031NL_ST25{9969840} (1)

<210> 200 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Integration of reporter construct into NR2F2 - 5' junction forward primer <400> 200 gcggaccact ttcatgctga ttc 23 <21e> 201 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Integration of reporter construct into NR2F2 - 5' junction reverse primer <400> 201 ctcctcgccc ttgctcacca t 21 <21e> 202 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Integration of reporter construct into NR2F2 - 3' junction forward primer <400> 202 cggcagttgg gattcgtgaa ttg 23 <21e> 203 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Integration of reporter construct into NR2F2 - 3' junction reverse primer <400> 203 caggatgtta atccacggag ggtc 24

Pagina 180

Claims

-86 - CONCLUSIONS

A method of integrating an exogenous nucleic acid sequence into the genome of a cell, the method comprising: a. providing a cell comprising a landing site in its genome, the landing site, in the direction of & to 3, successively comprising: a first recombination site, and a nucleic acid sequence encoding a selectable marker gene lacking a start codon; b. introducing into the cell an integration vector, the vector comprising (i) an exogenous nucleic acid sequence, and (ii) a construct comprising, in the 5' to 3' direction, sequentially: a promoter for directing expression of the selectable marker gene, a start codon, and a second recombination site; c. performing a recombination between the first and second recombination sites.

The method of claim 1, wherein the landing site comprises a first spacer located between the first recombination site and the nucleic acid sequence encoding a selectable marker gene lacking a start codon.

The method of claim 2, wherein the first spacer comprises less than 20 nucleotides, preferably comprises less than 15 nucleotides, even more preferably comprises less than 10 nucleotides, and most preferably comprises between 1 and 5 nucleotides.

The method of claims 2 or 3, wherein the first spacer consists of the sequence CATAT (SEQ ID NO: 1).

The method of claims 2 or 3, wherein the first spacer comprises a sequence of N(Y)x, wherein N is a nucleotide, and (Y) represents additional nucleotides, wherein 'x' represents the number of additional nucleotides, and where x is a multiple of 3.

-87 -

The method of claim 5, wherein the first spacer consists of the sequence GCGC (SEQ ID NO:2).

The method of any preceding claim, wherein the integration vector comprises a second spacer located between the start codon and the second recombination site.

The method of claim 7, wherein the second spacer comprises less than 20 nucleotides, preferably comprises less than 15 nucleotides, even more preferably comprises less than 10 nucleotides, and most preferably comprises between 1 and 5 nucleotides.

The method of claim 7 or 8, wherein the total number of nucleotides contained in the first and second spacers is a multiple of 3.

The method of claim 9, wherein the second spacer consists of the nucleotide G.

The method of claims 7 to 9, wherein the second spacer comprises a sequence of (Y)xNN(Y)x, wherein N is a nucleotide, and wherein (Y) represents additional nucleotides, wherein 'x' represents the number of additional nucleotides, and where x is a multiple of 3.

The method of claim 11, wherein the second spacer consists of the sequence GC.

The method of claim 7, wherein the first spacer consists of the sequence CATAT (SEQ ID NO: 1), and the second spacer consists of the nucleotide G.

The method of claim 7, wherein the first spacer consists of the sequence CATAT (SEQ ID NO: 1), and the second spacer consists of the nucleotide GC.

The method of any preceding claim, wherein the first recombination site is attP, and the second recombination site is attB, or wherein the first recombination site is attB, and the second recombination site is attP.

-88-

A method according to any one of the preceding claims, wherein the first recombination step is performed by first a recombinase enzyme, preferably wherein the first recombinase enzyme is a serine recombinase, preferably BxB1 or PhiC31.

A method according to any one of the preceding claims, wherein the method further comprises a second recombination step, preferably for removing secondary nucleic acid sequences from the genome of the cell.

The method of claim 17, wherein the landing site comprises a third recombination site and a fourth recombination site, and wherein the integration vector comprises a fifth recombination site and a sixth recombination site.

The method of claim 18, wherein the method comprises a step of performing a recombination between the third and sixth recombination sites, and performing a recombination between the fourth and fifth recombination sites.

A method according to any one of claims 17 to 19, wherein the second recombination step is performed by a second and optionally a third recombinase enzyme, preferably wherein the second and optionally the third recombinase is or are a tyrosine recombinase, for example a Cre or a Flp recombinase.

A method of identifying a cell that has undergone a successful recombination event, the method comprising: a. performing a method according to any one of claims 1 to 20, on one or more cells; b. identifying and optionally selecting a cell expressing the selectable marker gene.

A method of producing a population of cells each comprising an exogenous nucleic acid sequence successfully integrated into the respective genome, the method comprising:

-89- a. performing a method according to any one of claims 1 to 20, on a plurality of cells; b. identifying and optionally selecting the cells expressing the selectable marker; c. enriching the cells expressing the selectable marker.

A cell produced by a method according to any one of claims 1 to 20.

A population of cells produced by a method according to claim 22.

A cell comprising an exogenous nucleic acid sequence successfully integrated into its genome, the cell comprising a construct comprising, in the 5' to 3' direction, sequentially: a promoter for directing expression of a selectable marker gene, a start codon, a recombination scar, and a nucleic acid sequence encoding the selectable marker gene.

A nucleic acid sequence encoding a landing site, sequentially, in the 5 to 3 direction, comprising: a recombination site, and a nucleic acid sequence encoding a selectable marker gene lacking a start codon.

2/7. Landing site vector comprising a nucleic acid sequence according to claim 26.

28. Cell comprising a landing site in its genome, the landing site comprising, in the 5 to 3' direction, in sequence: a recombination site, and a nucleic acid sequence encoding a selectable marker gene lacking a start codon.

29. Nucleic acid sequence encoding an integration construct, wherein the integration construct comprises sequentially in the 5 to 3' direction: a promoter for directing expression of a selectable

-90 - marker gene, a start codon, and a recombination site, wherein the integration construct lacks a nucleic acid sequence encoding the selectable marker gene.

An integration vector comprising (i) an exogenous nucleic acid sequence, and (ii) an integration construct according to claim 29.

A cell comprising (i) a landing site in its genome, the landing site comprising, in the 5' to 3' direction, sequentially: a first recombination site, and a nucleic acid sequence encoding a selectable marker gene lacking a start codon; and (ii) an integration vector, the vector comprising (i) an exogenous nucleic acid sequence, and (ii) a promoter for directing expression of the selectable marker gene, a start codon, and a second recombination site.

A kit comprising (i) a nucleic acid sequence according to claim 26, or a landing site vector according to claim 27, or a cell according to claim 28; (ii) a nucleic acid sequence according to claim 29, or an integration vector according to claim 30; (ii) a recombinase enzyme or a nucleic acid sequence encoding a recombinase enzyme; and (iv) instructions for performing a method according to any one of claims 1 to 20.

The library of cells of claim 23, 24, or 25, wherein each cell comprises a different exogenous nucleic acid sequence.