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CN109957569A - Base editing system and method based on CPF1 albumen - Google Patents

Base editing system and method based on CPF1 albumen Download PDF

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Publication number
CN109957569A
CN109957569A CN201811578853.8A CN201811578853A CN109957569A CN 109957569 A CN109957569 A CN 109957569A CN 201811578853 A CN201811578853 A CN 201811578853A CN 109957569 A CN109957569 A CN 109957569A
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CN109957569B (en
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高彩霞
王延鹏
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Suzhou Qihe Biotechnology Co ltd
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Institute of Genetics and Developmental Biology of CAS
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Abstract

The present invention relates to genetic engineering fields.Specifically, the present invention relates to a kind of base edit methods based on CPF1 albumen.More specifically, the present invention relates to a kind of methods that Cpf1- deamination enzyme fusion proteins instructed by guide RNA carry out efficient base editor to the target sequence in organism (such as plant) genome, and the genetically modified organism (such as plant) and its offspring generated by the method.

Description

Base editing system and method based on CPF1 albumen
Technical field
The present invention relates to genetic engineering fields.Specifically, the present invention relates to a kind of base editors based on CPF1 albumen System and method.More particularly it relates to which a kind of Cpf1- deamination enzyme fusion proteins instructed by guide RNA are to life The system and method that target sequence in object (such as plant) genome carries out efficient base editor, and produced by the method Raw genetically modified organism (such as plant) and its offspring.
Background technique
Efficient crop improvement premise is that obtaining new genetic mutation, these mutation can be easily introduced into the modern times In cultivar.Genetic research, be based especially on full-length genome it is relevant studies have shown that the change of mononucleotide be constitute make physical property The main reason for shape difference.The variation of single base will lead to amino acid substitution, so as to cause superior allelic and excellent character Evolution.Before genome editor occurs, directional induction genome abrupt local (TILLING) be can be used as generating crop In improvement there is an urgent need to mutation method.However, the TILLING screening point mutation that takes time and effort, and identified often by The limitation of number and type.Genome editing technique, the genome editing technique for being based particularly on CRISPR/Cas9 system can be with The DNA mediated by homologous recombination (HR) repairs approach to realize the replacement for introducing particular bases in genomic locus.But mesh Before, the successful use of this method is very limited, and mainly occurs due to the double-strand chain rupture reparation of the HR mediation in plant Frequency is very low.In addition, effectively providing enough DNA recovery templates is also a current big difficulty.These problems make at present Rite-directed mutagenesis efficiently is simply implemented as a major challenge by way of HR in plant.
In recent years, using the binding characteristic of Cas9 and DNA and DNA deamination enzyme viability, Cas9 is merged with deaminase, it can To realize that the accurately transformation to target gene progress single base cytimidine (C) to thymidine (T) and adenine (A) are fast to bird The transformation of purine (G).Currently, mainly including SpnCas9-BE3, SpnCas9-AID and Cas9 for the system of the transformation of C to T The fusions of variant such as VQR-BE3, EQR-BE3 and VRER-BE3, in addition there are SaCas9-BE3 and variant SaKKH-BE3, The PAM limitation for making cytimidine (C) to thymidine (T) transformation of these combinations reduces and the range of editor more may be used Become.In addition, by way of artificial evolution, having developed can act on ssDNA's in the laboratory Harvard University David Liu in the recent period Adenine deaminase merges the Cas9-ABE system that may be implemented to carry out DNA the transformation of A to G with Cas9, compiles single base The functional orientation further expansion collected.It is current although these researchs make the single base editor of DNA have very big use scope Single base editing technique there are still many problems: first, be generally limited to due to the PAM of Cas9 and Cas9 variant rich in G/C Region, there is still a need for widen for the PAM type of single alkali editing system；Second, due to Cas9 and specific poor, the single base of editor itself There is still a need for raisings in terms of specificity for editing system；Third, due to the nCas9-BE3 and its BE3 of variant, there are also nCas9-ABE Single-stranded incise usually is generated on the non-targeted chain of target site, it is easy to prominent in generation single base during mispairing is repaired Also the certain of DNA and insertion can be generated while change, so also to be improved in terms of the high-fidelity of single base.Therefore, ability Still there is an urgent need to the new system and method for carrying out base editor to Plant Genome in domain.
Attached drawing description
The optimization for the Plant Genome cleavage activity that Fig. 1 .CPF1 is mediated.
The mutation for Plant Genome C to the T that Fig. 2 .CPF1 is mediated.
The mutation for Plant Genome A to the G that Fig. 3 .CPF1 is mediated.
Fig. 4 using CPF1 RNA cleavage activity carry out multidigit point while base editor.
Summary of the invention
One, it defines
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue Culture, microbiology, immunology relational language and laboratory operation step be in corresponding field widely used term and often Advise step.For example, standard recombinant dna and molecule clone technology used in the present invention is known to those skilled in the art, and It is more fully described below in the following literature: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning:A Laboratory Manual；Cold Spring Harbor Laboratory Press:Cold Spring Harbor, 1989 (hereinafter referred to as " Sambrook ").Meanwhile for a better understanding of the present invention, relational language is provided below Definition and explanation.
" Cpf1 nuclease ", " Cpf1 albumen " and " Cpf1 " is used interchangeably herein, and refers to including Cpf1 albumen Or the nuclease of the RNA guidance of its segment.Cpf1 is the component of CRISPR-Cpf1 genome editing system, can be in guide RNA (crRNA) targeting and cutting DNA target sequence form DNA double chain and are broken (DSB) under guidance.It include a DNA on Cpf1 albumen Cutting domain and an independent RNA cutting domain.The RNA cutting domain of Cpf1 albumen can process pre- CrRNA is to form mature crRNA.
" guide RNA " and " gRNA " are used interchangeably herein.The guide RNA for the genome editing system that Cpf1 is mediated Usually only be made of mature crRNA molecule, the sequence that wherein crRNA includes and target sequence have enough phase same sexes so as to target sequence The complementary sequence hybridization of column and instruct compound (Cpf1+crRNA) in conjunction with the target sequence sequence-specific.
" deaminase " refers to the enzyme of catalysis desamination reaction.In some embodiments of the present invention, what the deaminase referred to It is cytosine deaminase, deamination turns to uracil or Brdurd respectively for catalysis cytidine or deoxycytidine.In the present invention one In a little embodiments, the deaminase refers to adenine deaminase, can be catalyzed adenosine or desoxyadenossine (A) forms inosine (I)。
" genome " not only covers the chromosomal DNA being present in nucleus when for plant cell, but also including The organelle DNA being present in the subcellular components (such as mitochondria, plastid) of cell.
As used herein, " organism " includes any organism suitable for genome editor, preferably eucaryote.Organism Example include but is not limited to mammal such as people, mouse, rat, monkey, dog, pig, sheep, ox, cat；Poultry such as chicken, duck, goose；It plants Object includes monocotyledon and dicotyledon, such as rice, corn, wheat, sorghum, barley, soybean, peanut, arabidopsis etc..
" genetically modified organism " or " genetically modified cell " means in its genome comprising external source multicore The organism of the gene or expression regulation sequence of thuja acid or modification or cell.Such as exogenous polynucleotide can be steadily integrated into In the genome of organism or cell, and heredity is continuous from generation to generation.Exogenous polynucleotide can be either individually or as recombinant DNA construction The thin consolidation of body is into genome.The gene or expression regulation sequence of modification are the sequence described in organism or cellular genome Column replace comprising single or multiple deoxynucleotides, lack and add.For example, the genetically modified life obtained through the invention Object can include one or more C to T or A to G relative to wild type (organism accordingly without the genetic modification) Substitution.
" external source " for sequence means the sequence from alien species, or if coming from same species, refers to It is made up of premeditated human intervention from its native form and/or the sequence of locus significantly changed.
" polynucleotides ", " nucleic acid sequence ", " nucleotide sequence " or " nucleic acid fragment " be used interchangeably and be it is single-stranded or Double-stranded RNA or DNA polymer, optionally containing nucleotide base that is synthesis, non-natural or changing.Nucleotide is by such as Descend their single letter title to refer to: " A " is adenosine or desoxyadenossine (respectively corresponding RNA or DNA), and " C " indicates cytidine Or deoxycytidine, " G " indicate guanosine or deoxyguanosine, " U " indicates uridine, and " T " indicates deoxythymidine, " R " indicate purine (A or G), " Y " indicates pyrimidine (C or T), and " K " indicates that G or T, " H " indicate A or C or T, and " I " indicates inosine, and " N " indicates any Nucleotide.
" polypeptide ", " peptide " and " protein " is used interchangeably in the present invention, refers to the polymer of amino acid residue.The art Language is suitable for the ammonia that wherein one or more amino acid residues are the artificial chemical analogues of corresponding naturally occurring amino acid Base acid polymer, and it is suitable for naturally occurring amino acid polymer.Term " polypeptide ", " peptide ", " amino acid sequence " and " egg White matter " may also include modified forms, including but not limited to glycosylation, lipid connection, sulfation, glutaminic acid residue γ carboxylic Change, hydroxylation and ADP- ribosylation.
As used in the present invention, " expression construct ", which refers to, is listed in the load expressed in organism suitable for interested nucleotides sequence Body such as recombinant vector." expression " refers to the generation of function product.For example, the expression of nucleotide sequence can refer to the transcription of nucleotide sequence (as transcription generates mRNA or function RNA) and/or RNA translate into precursor or mature protein.
" expression construct " of the invention can be linear nucleic acid fragment, cyclic plasmid, viral vectors, alternatively, one In a little embodiments, the RNA (such as mRNA) that can be translated can be.
" expression construct " of the invention may include the regulating and controlling sequence and interested nucleotide sequence or phase of separate sources With source but by be different from it is usually naturally occurring in a manner of the regulating and controlling sequence and interested nucleotide sequence that arrange.
" regulating and controlling sequence " and " controlling element " is used interchangeably, refer to the upstream (5 ' non-coding sequence) positioned at coded sequence, Intermediate or downstream (3 ' non-coding sequence), and influence the transcription of related coding sequences, RNA processing or stability or translation Nucleotide sequence.
Regulating and controlling sequence may include but be not limited to promoter, translation leader sequence, introne and polyadenylation identification sequence.
" promoter " refers to control the nucleic acid fragment of another nucleic acid fragment transcription.In some embodiments of the invention In, promoter is can to control the promoter of genetic transcription in biological cell, whether is it deriving from the organism.It opens Mover can be constitutive promoter or tissue-specific promoter or developmental regulation promoter or inducible promoter.
" constitutive promoter " refers to the starting that will generally gene be caused to be expressed in most cases in most cell types Son." tissue-specific promoter " and " tissue-preferred promoter " is used interchangeably, and refers to main but nonessential exclusively exist The promoter expressing, and can be expressed in a kind of specific cells or cellular type in a kind of tissue or organ." developmental regulation opens Mover " refers to the promoter that its activity is determined by development event." inducible promoter " responds endogenous or external source sexual stimulus (ring Border, hormone, chemical signal etc.) and selective expression can manipulate the DNA sequence dna of connection.
As used herein, term " being operably connected " refer to controlling element (such as, but not limited to, promoter sequence, turn Record termination sequence etc.) it is connect with nucleic acid sequence (for example, coded sequence or opening code-reading frame), so that the transcription quilt of nucleotide sequence The transcriptional regulatory element control and adjusting.For controlling element region to be operably connected to the technology of nucleic acid molecules for this Known to field.
Nucleic acid molecules (such as plasmid, linear nucleic acid fragment, RNA etc.) or protein " importing " organism are referred to described in use Nucleic acid or protein inverting biological body cell, so that the nucleic acid or protein can function in cell.Institute of the present invention " conversion " includes stable conversion and instantaneous conversion.
" stable conversion ", which refers to, in exogenous nucleotide sequence quiding gene group, will cause foreign gene to stablize heredity.Once steady Fixed conversion, exogenous nucleic acid sequences are steadily integrated into the genome in the organism and its any successive generation.
" instantaneous conversion ", which refers to, imports nucleic acid molecules or protein in cell, executes function without foreign gene and stablizes something lost It passes.In instantaneous conversion, exogenous nucleic acid sequences unconformity is into genome.
As it is used herein, term " plant " includes entire plant and spawn, the cell of plant, tissue or portion Point.Term " plant part " includes any part of plant, including, such as, but not limited to: seed (including mature seed, do not have The immature embryo and premature seed of kind skin)；Plant cutting (plant cutting)；Plant cell；Culture plant cell Object；Plant organ (for example, pollen, embryo, flower, fruit, bud, leaf, root, stem, and related explant).Plant tissue or plant organ Can be seed, callus or any other be organized into the plant cell group of structure or function unit.Plant cell Or tissue culture can regenerate the physiology of the plant with the cell or tissue institute source and the plant of morphological feature, And the plant that there is substantially the same genotype with the plant can be regenerated.In contrast, some plant cells can not be again Produce plant.Regenerable cell in plant cell or tissue culture can be embryo, protoplast, meristematic cell, callus Tissue, pollen, leaf, anther, root, the tip of a root, silk, flower, kernel, fringe, cob, shell or stem.
Plant part includes the part of the part that can be harvested and the plant that can be used for raising up seed.It can be used for the plant portion bred Divide and include, such as, but not limited to: seed；Fruit；Cutting；Seedling；Stem tuber；And stock.The part that harvests of plant can be plant Any useful part, including, such as, but not limited to: flower；Pollen；Seedling；Stem tuber；Leaf；Stem；Fruit；Seed；And root.
Plant cell is the structure and physiology unit of plant.As it is used herein, plant cell include protoplast and Protoplast with part cell wall.Plant cell may be at the form (example of isolated individual cells or cell aggregation Such as, the cell of loose callus and culture), and can be more advanced organizational unit (for example, plant tissue, plant organ, And plant) a part.Therefore, plant cell can be protoplast, generate the cell of gamete, or can regenerate complete The cell of plant or the set of cell.Therefore, in embodiments herein, become comprising multiple plant cells and capable of regenerating The seed of whole plant is considered as a kind of " plant part ".
As it is used herein, term " protoplast " refers to cell wall by completely or partially removing, its lipid bilayer Exposed plant cell.Typically, protoplast is the separating plant cell of not cell wall, has regeneration cell culture The potentiality of object or whole plant.
Plant " offspring " includes any subsequent generation of plant.
" character " refers to physiological, form, biochemical or physics the feature of plant or specified plant material or cell.? In some embodiments, these features can be macroscopic, such as seed, size of plant etc.；Biochemistry skill can be used Art measurement index, such as in seeds or leaves albumen, starch or oil content content；The metabolism or physiology course of observable, such as Measure the resistance to water stress, specific salts, sugar or nitrogen concentration；Detectable gene expression dose；Or observable osmotic stress Resistance or the economical characters such as yield.In some embodiments, character further includes the ploidy (ploidy) of plant, such as to plant The important haploidy of object breeding (h kowtows loidy).In some embodiments, character further includes resistance of the plant to herbicide.
" economical character " is measurable index parameter, including but not limited to: leaf green, grain yield, growth rate, Fresh weight when total biomass or accumulation rate, maturation, it is mature when dry weight, fruit yield, seed production, plant total nitrogen content, Fruit nitrogen content, seed nitrogen content, plant nutrient tissue nitrogen content, plant total free amino acid content, fruit free amino acid Content, Seed Free Amino Acid content, plant nutrient tissue free aminoacid content, plant total protein content, fruit albumen contain Amount, seed protein, plant nutrient tissue protein content, drought resistance, the absorption of nitrogen, the lodging of root, harvest index, stem Lodging, plant height, fringe height, spike length, disease resistance, winter resistance, salt-resistance and tiller number etc..
Two, based on the base editing system of Cpf1 albumen
The present invention provides it is a kind of for in organism genome target sequence carry out base editor system, it includes It is following i) at least one of v):
I) base editor fusion protein and guide RNA；
Ii) the expression construct and guide RNA of the nucleotide sequence comprising encoding base editor fusion protein；
Iii) base editor fusion protein, and the expression construct of the nucleotide sequence comprising encoding guide RNA；
Iv) the expression construct of the nucleotide sequence comprising encoding base editor fusion protein, and comprising encoding guide RNA Nucleotide sequence expression construct；
V) expression of the nucleotide sequence of the nucleotide sequence comprising encoding base editor fusion protein and coding guide RNA Construct；
Wherein the base editor fusion protein includes the Cpf1 and deaminase of DNA cleavage activity missing, the guide RNA Can by the target sequence in the base editor fusion protein target gene group, cause in the target sequence one or more C to T or The substitution of person A to G.
Cpf1 includes a DNA cutting domain (RuvC), and after being mutated the DNA cleavage activity of Cpf1 can lack It loses, forms " Cpf1 of DNA cleavage activity missing ".The Cpf1 of the DNA cleavage activity missing still retains the DNA of gRNA guidance Binding ability.Therefore, in principle, when with other protein fusion, the Cpf1 of DNA cleavage activity missing can be simply by With suitable guide RNA co-express and by the substantially any DNA sequence dna of other targeting proteins.
The Cpf1 of DNA cleavage activity missing of the present invention can be derived from the Cpf1 of different plant species, for example, being derived from Francisella novicida U112, Acidaminococcussp.BV3L6 and Lachnospiraceae bacterium ND2006 to be referred to as FnCpf1 (such as the amino acid sequence of wild type is shown in SEQ ID NO:19), AsCpf1 (such as wild The amino acid sequence of raw type is shown in SEQ ID NO:18) and the Cpf1 albumen of LbCpf1 (such as the amino acid sequence of wild type shows In SEQ ID NO:20).
In some embodiments, the Cpf1 of the DNA cleavage activity missing is the FnCpf1 of DNA cleavage activity missing. In some embodiments, the FnCpf1 of the DNA cleavage activity missing includes D917A relative to wild type FnCpf1 Mutation.
In some embodiments, the Cpf1 of the DNA cleavage activity missing is the AsCpf1 of DNA cleavage activity missing. In some embodiments, the AsCpf1 of the DNA cleavage activity missing includes D908A relative to wild type AsCpf1 Mutation.
In some preferred embodiments, the Cpf1 of the DNA cleavage activity missing is DNA cleavage activity missing LbCpf1.In some embodiments, the LbCpf1 of the DNA cleavage activity missing includes relative to wild type LbCpf1 D832A mutation.
In some embodiments, the Cpf1 of the DNA cleavage activity missing retains its RNA cleavage activity, so as to right Pre-crRNA is processed to form mature crRNA.Therefore, in some embodiments, include volume in system of the invention The expression construct of the nucleotide sequence of code guide RNA may include concatenated multiple and different guide RNA (crRNA) precursors of coding Sequence, after transcription can by the DNA cleavage activity lack Cpf1 process to form multiple and different guide RNA (crRNA), to target multiple and different target sequences simultaneously.
In some embodiments of the invention, the deaminase in fusion protein is cytidine deaminase, such as carries rouge egg White B mRNA edits complex (APOBEC) family deaminase.
Cytidine deaminase can act on forming uracil (U) with the deaminizing of cytidine on catalytic dna (C).The present inventor makes us It has surprisingly been found that the Cpf1 that DNA cleavage activity lacks is merged with cytidine deaminase, under the guidance of guide RNA, fusion protein Can be with the target sequence in target gene group, since the DNA cleavage activity of Cpf1 inactivates missing, DNA double chain is not cut, and is melted Guide's Cpf1- RNA-DNA compound can be formed the cytidine deamination of the single stranded DNA of middle generation by the cytidine deaminase in hop protein It is converted into U, then realizes the substitution of C to T by base mispairing reparation.
Cytidine deaminase of the present invention can especially receive cytidine deaminase of the single stranded DNA as substrate.The present invention The example of available cytidine deaminase includes but is not limited to: APOBEC1 deaminase, activation-inducing cytidine deaminase (AID), APOBEC3G or CDA1.In certain specific embodiments of the invention, the cytidine deaminase includes shown in SEQ ID NO:1 Amino acid sequence.
In the case that deaminase in the fusion protein is cytidine deaminase, base editing system of the invention can be by base Because one or more C in group target sequence sport T, also referred to as Cpf1-PBE system.
In cell, uracil dna glycosylase catalysis U from the removal on DNA and starts base excision repair (BER), Cause to repair U:G at C:G.Therefore, without being bound by any theory, in base editor fusion protein of the invention or of the invention The efficiency of base editor will be increased in system comprising uracil dna glycosylase inhibitor.
Therefore, in some embodiments for being related to Cpf1-PBE system of the invention, the base editor fusion protein Also include uracil dna glycosylase inhibitor (UGI).In some embodiments, the uracil dna glycosylase Inhibitor includes amino acid sequence shown in SEQ ID NO:2.
In some embodiments of the invention, the deaminase is adenine deaminase.
Naturally occurring adenine deaminase is turned the adenosine on single stranded RNA by deamination with often using RNA as substrate Become inosine (I).Recently, by the method for directed evolution, the tRNA adenine deaminase TadA for having been based on Escherichia coli is obtained The deoxyguanosine on single stranded DNA can be changed into the DNA dependent form adenine of inosine (I) using single stranded DNA as substrate by obtaining Deaminase.Referring to Nicloe M.Gaudelli et al., doi:10.1038/nature24644,2017.
The present inventor is it has surprisingly been found that the Cpf1 and DNA dependent form adenine deaminase that DNA cleavage activity is lacked Fusion, under the guidance of guide RNA, fusion protein can be with the target sequence in targeted plants genome, since the DNA of Cpf1 is cut Activity missing, DNA double chain are not cut, and the DNA dependent form adenine deaminase in fusion protein can be by guide Cpf1- The adenosine deamination that RNA-DNA compound forms the single stranded DNA of middle generation is converted into inosine (I), since archaeal dna polymerase can be by inosine (I) it is handled as guanine (G), therefore the substitution of A to G may be implemented by base mispairing reparation.Therefore, in the fusion protein Deaminase be DNA dependent form adenine deaminase in the case where, base editing system of the invention can be by genome target sequence One or more A in column sport G, also referred to as Cpf1-ABE system.
In some embodiments of the present invention, the DNA dependent form adenine deaminase is that Escherichia coli tRNA gland is fast The variant of purine deaminase TadA (ecTadA), can especially receive variant of the single stranded DNA as substrate, and the variant is opposite Include one or more groups of mutation selected from the following in wild type ecTadA:
1) A106V and D108N；
2) D147Y and E155V；
3) L84F, H123Y and I156F；
4)A142N；
5) H36L, R51L, S146C and K157N；
6)P48S/T/A；
7)A142N；
8)W23L/R；
9)R152H/P。
In the embodiment of the invention, the DNA dependent form adenine deaminase (ABE version 7.9) relative to Wild type ecTadA include following mutation: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, A142N, D147Y, E155V and R152P.
In the embodiment of the invention, the DNA dependent form adenine deaminase (ABE version 7.10) relative to Wild type ecTadA include following mutation: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, D147Y, E155V and R152P.
Wild type ecTadA amino acid sequence is as follows: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVH NNRVIGEGWNR PIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIH SRIGRVVFGARDAK TGAAGSLMDVLHHPGMNHRVEITEGILADECAALL SDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO:3).One In a little embodiments, wherein initial methionine can be not present.
The following institute of DNA dependent form adenine deaminase (ABE version 7.10) amino acid sequence derived from preferred ecTadA Show: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR AIGLHDPTAHAEIMALRQGGLVMQ NYRLIDATLYVTFEPCVMCAGAMIH SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALL CYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:4).In some embodiments, wherein initial methionine can not be deposited ?.
In some embodiments of the present invention, the deaminase is fused the Cpf1 lacked to the DNA cleavage activity N-terminal.In some embodiments, the deaminase is fused the C-terminal of the Cpf1 lacked to the DNA cleavage activity.
In some preferred embodiments, the N-terminal fusion of the DNA dependent form adenine deaminase has corresponding wild type Adenine deaminase.It is expected that DNA dependent form adenine deaminase, which forms heterodimer with wild type adenine deaminase, to be shown Write the editor's activity for improving fusion protein A to G.
In some embodiments of the present invention, the deaminase and the Cpf1 of DNA cleavage activity missing are by connecing Head fusion.The connector can be long 1-50 (such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18,19,20 or 20-25,25-50) or more amino acid, the non-functional amino acid sequence without the above structure of second level Column.For example, the connector can be flexible joint, such as GGGGS, GS, GAP, (GGGGS) x 3, GGS and (GGS) x7 etc..? In some specific embodiments, the connector is XTEN connector.In some specific embodiments, long 32 ammonia of the connector Base acid.In some specific embodiments, the amino acid sequence of the connector are as follows: SGGSSGGSSGSETPGTSESATPES SGGSSGGS。
In some embodiments of the present invention, base editor's fusion protein of the invention also includes nuclear localization sequence (NLS).In general, one or more NLS in the base editor fusion protein should have enough intensity, to plant The base editor fusion protein is driven in the core of object cell so that the accumulation of the amount of its base editting function can be achieved.In general, The active intensity of nuclear location is specific by the number of NLS, position, used one or more in the base editor fusion protein NLS or these factors combination determine.
In some embodiments of the present invention, the NLS of base editor's fusion protein of the invention can be located at N-terminal and/ Or C-terminal.In some embodiments, the base editor fusion protein includes about 1,2,3,4,5,6,7,8,9,10 or more Multiple NLS.In some embodiments, the base editor fusion protein be included in or close to N-terminal about 1,2,3,4,5, 6,7,8,9,10 or more NLS.In some embodiments, the base editor fusion protein is included in or close to C About 1,2,3,4,5,6,7,8,9,10, end or more NLS.In some embodiments, the base editor fusion protein packet Containing these combination, it is such as included in one or more NLS of N-terminal and one or more NLS in C-terminal.It is more than one when existing When a NLS, each can be selected as independent of other NLS.In some preferred embodiments of the invention, the alkali Base editor's fusion protein includes 2 NLS, such as 2 NLS are located at N-terminal and C-terminal.
In general, NLS is short by the positively charged lysine being exposed on protein surface or arginic one or more Sequence composition, but other kinds of NLS is also known.The non-limiting example of NLS includes: KKRKV (nucleotide sequence 5 '- AAGAAGAGAAAGGTC-3 '), PKKKRKV (nucleotide sequence 5 '-CCCAAGAAGAAGAGGAAGGTG-3 ' or ) or SGGSPKKKRKV (5 '-TCGGGGGGGAGCCCAAAGAAGAAGC of nucleotide sequence CCAAAGAAGAAGAGGAAGGTT GGAAGGTG-3’)。
In certain embodiments of the present invention, the N-terminal of the base editor fusion protein includes shown in PKKKRKV The NLS of amino acid sequence.In certain embodiments of the present invention, the C-terminal of the base editor fusion protein includes The NLS of amino acid sequence shown in SGGSPKKKRKV or KRPAATKKAGQAKKKK.
In addition, base editor fusion protein of the invention can also include others according to the position DNA of required editor Positioning sequence, such as cytoplasm positioning sequence, chloroplast targeting sequence, mitochondria positioning sequence etc..
In some embodiments for being related to Cpf1-PBE system of the invention, the base editor fusion protein also includes Uracil dna glycosylase inhibitor (UGI), and also in the N-terminal or C-terminal of the UGI immediately two NLS.In some preferred realities It applies in scheme, base editor's fusion protein of the invention includes the amino acid sequence selected from SEQ ID NO:24-29.
In order to obtain effective expression, in certain embodiments of the present invention, the encoding base editor fusion protein Nucleotide sequence carries out codon optimization for the biological species of pending base editor.
Codon optimization refers to the password by more frequently or most frequently using in the gene of host cell Filial generation replaces at least one codon of native sequences (for example, about or more than about 1,2,3,4,5,10,15,20,25,50 or more Multiple codons maintain the natural acid sequence and modification of nucleic acids sequence simultaneously to enhance in host cell interested The method of expression.Different species show specific preference for certain codons of specific amino acids.Codon preference (difference that the codon between biology uses) is often related to the translation efficiency of mRNA (mRNA), and the translation efficiency Then it is considered the availability of the property and specific transfer RNA (tRNA) molecule dependent on the codon being translated.Intracellular choosing The advantage of fixed tRNA generally reflects the codon for being used most frequently for peptide synthesis.Therefore, gene can be customized to be based on close Best gene expression of the numeral optimization in given biology.Codon usage table can be readily available, such aswww.kazusa.orjp/codon/Upper obtainable codon uses in database (" Codon Usage Database "), and And these tables can be adjusted by different modes it is applicable.Referring to Nakamura Y. etc., " Codon usage tabulated From theinternational DNA sequencedatabases:statusfortheyear2000. Nucl.Acids Res., (2000) 28:292.
In some specific embodiments, base editor fusion protein of the invention is by being selected from SEQ ID NO:8-9,11- 12 or 14-15's is nucleotide sequence coded.
In some embodiments of the present invention, the nucleotide sequence of the encoding base editor fusion protein and/or described The nucleotide sequence of coding guide RNA is operably connected with expression regulation element such as promoter.
The example of promoter workable for the present invention includes but is not limited to that polymerase (pol) I, pol II or pol III are opened Mover.The example of pol I promoter includes chicken RNApol I promoter.The example of pol II promoter is including but not limited to big and small Early stage (CMV) promoter, the long end of Rous sarcoma virus repeat (RSV-LTR) promoter and simian virus 40 to cellular virus immediately (SV40) immediate early promoter.The example of pol III promoter includes U6 and H1 promoter.Inducible promoter can be used Such as metallothionein promoter.Other examples of promoter include T7 phage promoter, T3 phage promoter, beta galactose Glycosides enzyme promoters and Sp6 phage promoter.When be used for plant when, promoter can be cauliflower mosaic virus 35 S promoter, Maize Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, corn U3 promoter, rice actin promoters.
Preferably, guide RNA (crRNA) is expressed using Ubi-1 promoter and is cut into ribozyme such as HDV ribozyme It is ripe.
In one embodiment, an introne is added after Ubi-1 promoter can be enhanced destination protein or the table of RNA It reaches.
In some specific embodiments, the expression construct for expressing base fusion protein of the invention includes SEQ Expression cassette shown in ID NO:10 or 13.Or the expression construct includes expression regulation sequence shown in SEQ ID NO:30 Column.
Three, the method for genetically modified organism is generated
On the other hand, the present invention provides a kind of methods for generating genetically modified organism (such as plant), including It is used for of the invention to carry out biological cell described in the system introducing of base editor the target sequence in organism genome, by The base editor fusion protein is targeted the target sequence in the Plant Genome by this described guide RNA, leads to the target sequence One or more C in column are replaced by T or one or more A are replaced by G.
It can be encoded by the target sequence or crRNA that Cpf1 albumen and guide RNA (i.e. crRNA) compound identify and target The design of sequence is referred to such as Zhang et al., Cell 163,1-13, October 22,2015.In general, this Invention genome editing system targeting 5 ' end of target sequence need to include before between region sequence adjacent to motif (protospacer Adjacent motif) (PAM) 5 '-TTTN or 5 '-YTN, wherein N is selected from C and T independently selected from A, G, C and T, Y.
For example, in some embodiments of the present invention, the target sequence has a structure that 5 '-TTTN-NX- 3 ' or 5’-YTN-NX- 3 ', wherein N is selected from C and T independently selected from A, G, C and T, Y；X is the integer of 15≤X≤35；Nx indicates X Continuous nucleotide.
In the present invention, the target sequence of pending modification can be located at any position of genome, such as positioned at function base In cause such as protein coding gene, such as gene expression regulation area such as promoter region or Enhancer district can be located at, thus real The now modification to gene function modification or to gene expression.
A to G in the cell target sequence or C can be detected by T7EI, PCR/RE or sequencing approach to be compiled to T base Volume.
In the method for the invention, the system of the base editor can pass through various sides well known to those skilled in the art Method imports cell.Can be used for include but is not limited to by the method that genome editing system of the invention imports cell: calcium phosphate turns Dye, Protoplast fusion, electroporation, liposome transfection, microinjection, virus infection (such as baculoviral, vaccinia virus, adenovirus, gland Correlated virus, slow virus and other virus), particle bombardment, PEG mediate protoplast transformation, soil Agrobacterium mediate turn Change.
The cell that can carry out genome editor by means of the present invention can come from for example, mammal such as people, small Mouse, rat, monkey, dog, pig, sheep, ox, cat；Poultry such as chicken, duck, goose；Plant, including monocotyledon and dicotyledon, such as Rice, corn, wheat, sorghum, barley, soybean, peanut, arabidopsis etc..
Method of the invention is particularly suitable for generating genetically modified plant, such as crop plants.In production of the invention In the method for raw genetically modified plant, the base editing system can be led in various methods well known to those skilled in the art Enter plant.Can be used for include but is not limited to by the method that base editing system of the invention imports plant: particle bombardment, PEG are situated between Protoplast transformation, the conversion that soil Agrobacterium mediates, the conversion of plant virus mediation, pollen tube passage method and the ovary note led Penetrate method.
In the method for the genetically modified plant of generation of the invention, only need to import or generate in plant cell described in The modification to target sequence can be realized in base editor fusion protein and guide RNA, and the modification can stablize heredity, be not necessarily to By the base editing system stable conversion plant.This avoid the potential works that misses the target for the base editing system being stabilized With, also avoid the integration of exogenous nucleotide sequence in the plant genome, thus have higher biological safety.
In some preferred embodiments, described import under there is no selection pressure is carried out, to avoid exogenous nucleotide The integration of acid sequence in the plant genome.
In some embodiments, it is described importing include base editing system of the invention is converted it is thin to isolated plant Then born of the same parents or tissue make the transformed plant cell or regeneration full plants.Preferably, selection pressure is being not present The regeneration is carried out under power, that is to say, without using any for the selection base carried on expression vector in tissue culture procedures The selective agent of cause.The regeneration efficiency of plant can be improved without using selective agent, acquisition is without exogenous nucleotide sequence through modifying Plant.
In other embodiments, base editing system of the invention can be converted into the particular portion to full plants Position, such as blade, stem apex, pollen tube, young fringe or hypocotyl.This carries out the regenerated plant of tissue cultures particularly suitable for being difficult to Conversion.
In certain embodiments of the present invention, directly by the protein of vivoexpression and/or the RNA molecule of in-vitro transcription It converts to the plant.The protein and/or RNA molecule can realize base editor in plant cell, then by cell Degradation, avoids the integration of exogenous nucleotide sequence in the plant genome.
Can carry out the plant of base editor by means of the present invention includes monocotyledon and dicotyledon.Example Such as, the plant can be crop plants, for example, wheat, rice, corn and soybean, sunflower, sorghum, rape, clover, cotton, Barley, grain, sugarcane, tomato, tobacco, cassava or potato.
In certain embodiments of the present invention, wherein the target sequence is related to plant trait such as economical character, thus The base editor causes the plant to have the character changed relative to wild-type plant.
In the present invention, the target sequence of pending modification can be located at any position of genome, such as positioned at function base In cause such as protein coding gene, such as gene expression regulation area such as promoter region or Enhancer district can be located at, thus real The now modification to gene function modification or to gene expression.Correspondingly, in certain embodiments of the present invention, the C Substitution to T or A to G leads to the truncation (generating terminator codon) of amino acid substitution or target protein in target protein.At this In other embodiments of invention, the substitution of the C to T or A to G cause the expression of target gene to change.
In certain embodiments of the present invention, after the method also includes obtaining the genetically modified plant Generation.
On the other hand, the present invention also provides genetically modified plants or its offspring or part thereof, wherein the plant Above-mentioned method obtains object through the invention.
On the other hand, the present invention also provides a kind of plant breeding method, including by method above-mentioned through the invention The the first genetically modified plant obtained and the second plant hybridization for not containing the genetic modification, so that the heredity be repaired Decorations import the second plant.
Embodiment
Construct Ubi-CPF1-PBE/ABE expression vector
ABE, XTEN, dCPF1 sequence carry out codon optimization for plant and order from GenScript (Nanjing). Use primer pair HindIII-F (with HindIII restriction site) and EcoRI (there is EcoRI restriction site) amplification overall length DCPF1-ABE segment.PCR product is digested with HindIII and EcoRI, is inserted into the pJIT163- of both enzymic digestions GFP carrier (carrier sequence is shown in SEQ ID NO:16) is to generate fusion expression vector dCPF1-ABE.
PBE, XTEN, dCPF1 sequence carry out codon optimization for plant and order from GenScript (Nanjing). Use primer pair HindIII-F (with HindIII restriction site) and EcoRI (there is EcoRI restriction site) amplification overall length DCPF1-PBE segment.PCR product is digested with HindIII and EcoRI, is inserted into the pJIT163- of both enzymic digestions GFP carrier (carrier sequence is shown in SEQ ID NO:16) is to generate fusion expression vector dCPF1-PBE.
Construct sgRNA expression vector
According to description (Wang, Y.et al.Simultaneous editing of three homoeoalleles before in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat. Biotechnol.32,947-951,2014；Shan, Q.et al.Targeted genome modification of crop Plants using a CRISPR-Cas system.Nat.Biotechnol.31,686-688,2013；And Liang, Z.et al.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.J Genet Genomics.41,63-68,2014) it is based on pTaU6-sgRNA (Addgene ID53062) or pOsU3-sgRNA (Addgene ID53063) or pZmU3-sgRNA (Addgene ID5306) or OsU3/TaU6-tRNA-sgRNA (Zhang Et al. 2017.Genome Biology.DOI:10.1186/s13059-017-1325-9) building sgRNA expression vector.This Outside, start tup enzyme and crRNA also by II type promoter generate crRNA (Tang et al.Nature plant, Doi:10.1038/nplants.2017.18)
pUbi-mGFPP-crRNA、pUbi-DEP1-sgRNA、pUbi-DEP1-crRNA、pUbi -DME-crRNA.
BFP and GFP expression vector
PUbi-mGFP, the carrier sequence are shown in SEQ ID NO:17.
Protoplast measurement
Wheat Bobwhite kind, rice OryzasativaLcv.Nipponbare kind are used in our current research.The protoplast as described below that carries out turns Change.Average conversion efficiency is 55-70%.Every kind of plasmid is converted with 10 μ g by PEG mediated method, after 48 hours, is collected Protoplast extracts DNA and measures for T7EI and PCR-RE.
Wheat protoplast preparation and conversion
1) the tender blade of wheat is taken, intermediate portion is cut into the silk of 0.5-1mm, the Mannitol for being put into 0.6M is molten It is protected from light processing 10 minutes in liquid, then with strainer filtering, puts it into 50ml enzyme solution and is protected from light for 20-25 DEG C, 10rmp, which slowly rocks, to disappear Change 5 hours.
2) plus 10ml W5 dilutes enzymolysis product, filters enzymolysis liquid (50ml) in round bottom centrifuge tube with 75 μm of nylon leaching films.
3) 23 DEG C, 100g, it is centrifuged 3min, abandons supernatant.
4) it is gently hanged with W510ml, placing 30min on ice settles protoplast gradually, abandons supernatant.
5) plus appropriate MMG suspends, to be transformed as on ice.
6) add 10-20 μ g plasmid, 200 μ l protoplasts (about 4 × 10 in 2ml centrifuge tube5Cell), what 220 μ l newly matched PEG solution mixes, 10-20 minutes Induction Transformations of room temperature avoid light place.
7) slowly add 880 μ l W5 solution after Induction Transformation, be gently mixed by inversion, 100g horizontal centrifugal 3min, inhale and abandon Supernatant.
8) plus 2ml W5 solution is resuspended, and is transferred in six orifice plates, the culture of room temperature (or 25 DEG C) dark place.If primary for extracting Plastid genome DNA need to cultivate 48h.
Rice protoplast preparation and conversion:
1) seedling leaf sheath part separated protoplast is chosen, is cut into about 0.5mm wide with sharp cutter.
2) it is transferred in 0.6M Mannitol solution at once after cutting, avoid light place 10min.
3) Mannitol solution is filtered out, is transferred in enzymolysis liquid, is protected from light and vacuumizes 30min.
4) it is protected from light enzymatic hydrolysis 5-6h, while slowly shaking (decolorization swinging table, speed 10).
5) after digesting, isometric W5 is added, level shakes 10sec, discharges protoplast.
6) using 40 μm of nylon membrane filtering protoplasts to 50ml round bottom centrifuge tube, then plus W5 solution flushing.
7) 250g horizontal centrifugal 3min precipitates protoplast, inhales and abandons supernatant.
8) plus protoplast is resuspended in 10ml W5, and 250g is centrifuged 3min, abandons supernatant.
9) plus appropriate MMG solution resuspension protoplast concentration is 2 × 106/ml。
Note: all of above step is carried out in room temperature.
10) add 10-20 μ g plasmid, 200 μ l protoplasts (about 4 × 10 in 2ml centrifuge tube5Cell), 220 μ l newly match PEG solution, mix, 10-20 minutes Induction Transformations of room temperature avoid light place.
11) slowly add 880 μ l W5 solution after Induction Transformation, be gently mixed by inversion, 250g horizontal centrifugal 3min, inhale Abandon supernatant.
12) plus 2ml WI solution is resuspended, and is transferred in six orifice plates, the culture of room temperature (or 25 DEG C) dark place, if for extracting original Raw plastid genome DNA, need to cultivate 48h.
PCR/RE detection:
1) plant genome DNA is extracted.
2) gene specific primer is synthesized, expands the segment containing target site, length is between 350-1000bp:
10×EasyTaq Buffer 5μl
dNTP(2.5mM) 4μl
Forward primer (10 μM) 2μl
Forward primer (10 μM) 2μl
Easy Taq 0.5μl
DNA 2μl
ddH2O To 50 μ l
3) General reactions condition is: 94 DEG C of denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 30s expand Increase 30 to 35 circulations；72 DEG C of heat preservation 5min；12 DEG C of heat preservations.Take 5 μ l PCR product electrophoresis detections.
4) digestion with restriction enzyme PCR product, general digestion system are as follows:
10×Fastdigest Buffer 2ul
Restriction enzyme 1 μl
PCR product 3-5μl
ddH2O To 20 μ l
5) 37 DEG C, digestion 2-3h.The detection of 1.2% agarose gel electrophoresis.
6) the mutation band not cut in recovery purifying PCR product carries out TA clone.Reaction system is as follows:
pEasy-T Vector 1μl
The PCR product that do not cut of recycling 4μl
7) 22 DEG C of connection 10min, Transformed E .coli competent cell apply LB solid plate (Amp100, IPTG and X- Gal), 12-16h is cultivated, white colony identification positive colony is selected, send sequencing.
Deep sequencing
Different sgRNA expression vectors converts respectively with Ubi-CPF1-PBE/ABE expression vector to wheat, rice plastid After 48 hours, protoplast is collected, DNA is extracted and carries out deep sequencing.In first round PCR, target region uses locus specificity Primer is expanded.In two wheel PCR, forward and reverse label is added to PCR product end and carries out library construction.Merge etc. Measure different PCR products.Then sample uses Illumina High-Seq 4000 in Beijing Genomics Institute Sequencing.
Embodiment
The optimization for the Plant Genome cleavage activity that embodiment 1.CPF1 is mediated.
Editor activity difference in different articles of the CPF1 in plant cell is larger, and different types of CPF1 it Between cleavage activity difference it is also very big.
The present embodiment is optimized by the nuclear state that enters to AsCPF1, FnCPF1 and LbCPF1, while also to crRNA Promoter optimize, cleavage activity of the Lai Tigao CPF1 in plant cell.Construct AsCPF1, FnCPF1 and LbCPF1 1-4 NLS carrier, and construct U3/U6 and UBI starting the different carriers that crRNA is generated by ribozyme (such as Fig. 1).It can be worked by three kinds of CPF1 that the result of PCR/RE can be seen that 2 NLS, and LbCPF1 efficiency is higher (SEQ ID NO:5-7 is respectively the coded sequence of ASCPF1-2NLS, FNCPF1-2NLS and LBCPF1-2NLS, can be easy to obtain Obtain corresponding amino acid sequence).For the target site of this gene of OsPDS, it can be seen that the efficiency of 2NLS-LbCPF1 wants high In NLS-LbCPF1, higher than the construct of some other report.
The mutation (CPF1-PBE) for Plant Genome C to the T that embodiment 2.CPF1 is mediated
With reference to CPF1 the plant cell cleavage activity the characteristics of, following dCPF1-PBE system: dAsCPF 1- is constructed 2NLS-PBE,dFnCPF1-2NLS-PBE,dLbCPF1-2NLS-PBE.Wherein the NLS at the end C be individually placed to UGI one end and It has been individually placed to the both ends of UGI.CrRNA is started with UBI1 and is cut with ribozyme.Shown using PCR/RE testing result DFnCPf1 and dLbCPF1 detected editor's activity, and activity of the NLS in one end of UGI it is higher (SEQ ID NO:8,9 The coded sequence of dFNCPF1-PBE-2NLS and dLbCPF1-2NLS-PBE is shown respectively, corresponding amino acid sequence can be easy to get Column).In addition, also constructing the dCPF1-PBE2-X of strengthening version, i.e., it joined an introne behind with ZmUbi-1 promoter Come the expression that increases dCPF1-PBE, (SEQ ID NO:10 is shown comprising ZmUbi-1 promoter and intron sequences DLBCPF1-PBE-2NLS expression cassette).
The mutation (CPF1-ABE) for Plant Genome A to the G that embodiment 3.CPF1 is mediated
Construct following CPF1-ABE system: dAsCPF1-1NLS-ABE, dFnCPF1-NLS-ABE, dLbCPF1- 1NLS-ABE and dAsCPF1-2NLS-ABE, dFnCPF1-2NLS-ABE, dLbCPF1-2NLS-ABE, wherein ABE is wrapped again Include two versions of ABE7.9 and ABE7.10.CrRNA is started with UBI1 and is cut with ribozyme.
Using Fig. 3 E GFP base editor's reporting system the result shows that: dFnCPF1-ABE7.10 (SEQ ID NO: 11) it can work with dLbCPF1-ABE7.9 and dLbCPF1-ABE7.10 (SEQ ID NO:12), and 7.10 are higher than 7.9 (Fig. 3 F).
Show that dLbCPF1-ABE7.10 detected activity using PCR/RE testing result, and 2NLS is higher than 1NLS.In addition, also constructing the dCPF1-ABE2 of two strengthening versions, i.e., it joined an introne behind UBI1 promoter Increase the expression (dCPF1-ABE2-X1) (SEQ ID NO.13) of dCPF1-ABE, there are also by ABE building CPF1 C-terminal (dCPF1-ABE2-X2/X3) (SEQ ID NO.14,15) utilizes the result of GFP base editor's reporting system of Fig. 3 E: DCPF1-ABE2-X2/X3 edits activity and is higher than dLbCPF1-ABE7.10 (Fig. 3 G).
The gene editing optimization that embodiment 4.CPF1 is mediated
In order to continue to improve the editorial efficiency of CPF1, we continue to optimize CPF1 system, mediate first to CPF1 All expression vectors of editor started using BdUbi10 promoter, to increase its expression quantity.In addition, we are also right The starting of crRNA is started using II type promoter, and crRNAArray is put into the 5 ' UTR or 3 ' UTR regions of expressing gene, is come The editorial efficiency of CPF1 is improved by improving the expression of mRNA.
Correlated series description:
SEQ ID NO.1 cytidine deaminase amino acid sequence
SEQ ID NO.2 uracil dna glycosylase inhibitor (UGI) amino acid sequence
SEQ ID NO.3 wild type ecTadA amino acid sequence
DNA dependent form adenine deaminase (ABE version 7.10) amino acid sequence derived from SEQ ID NO.4 ecTadA
SEQ ID NO.5 ASCPF1-2NLS coded sequence
SEQ ID NO.6 FNCPF1-2NLS coded sequence
SEQ ID NO.7 LBCPF1-2NLS coded sequence
SEQ ID NO.8 dFNCPF1-PBE-2NLS coded sequence
SEQ ID NO.9 dLBCPF1-PBE-2NLS coded sequence
SEQ ID NO.10 promoter+introne+dLBCPF1-PBE-2NLS coded sequence
SEQ ID NO.11 dFNCPF1-ABE7.10-2NLS coded sequence
SEQ ID NO.12 dLBCPF1-ABE7.10-2NLS coded sequence
SEQ ID NO.13 promoter+introne+dLBCPF1-ABE2-x coded sequence
SEQ ID NO.14 LBCPF1-ABE2-x2 coded sequence
SEQ ID NO.15 LBCPF1-ABE2-x3 coded sequence
SEQ ID NO.16 PJIT163-GFP
SEQ ID NO:17 pBUI-mGFP
SEQ ID NO:18 ASCPF1 amino acid sequence
SEQ ID NO:19 FNCPF1 amino acid sequence
SEQ ID NO:20 LBCPF1 amino acid sequence
SEQ ID NO:21 ASCPF1-2NLS amino acid sequence
SEQ ID NO:22 FNCPF1-2NLS amino acid sequence
SEQ ID NO:23 LBCPF1-2NLS amino acid sequence
SEQ ID NO:24 dFNCPF1-PBE-2NLS amino acid sequence
SEQ ID NO:25 dLBCPF1-PBE-2NLS amino acid sequence
SEQ ID NO:26 dFNCPF1-ABE7.10-2NLS amino acid sequence
SEQ ID NO:27 dLBCPF1-ABE7.10-2NLS amino acid sequence
SEQ ID NO:28 LBCPF1-ABE2-x2 amino acid sequence
SEQ ID NO:29 LBCPF1-ABE2-x3 amino acid sequence
SEQ ID NO:30 promoter+introne nucleotide sequence.
Sequence table
<110>Inst. of Genetics and Development Biology, CAS
<120>based on the base editing system and method for CPF1 albumen
<130> 149132
<150> 201711403490.X
<151> 2017-12-22
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 227
<212> PRT
<213> Rattus norvegicus
<400> 1
Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg
1 5 10 15
Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg
20 25 30
Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser
35 40 45
Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn
50 55 60
Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg
65 70 75 80
Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser
85 90 95
Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe
100 105 110
Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln
115 120 125
Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr
130 135 140
Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro
145 150 155 160
Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu
165 170 175
Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu
180 185 190
Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala
195 200 205
Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala
210 215 220
Thr Gly Leu
225
<210> 2
<211> 87
<212> PRT
<213> Bacillus phage
<400> 2
Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
1 5 10 15
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile
20 25 30
Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu
35 40 45
Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
50 55 60
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile
65 70 75 80
Lys Met Leu Ser Gly Gly Ser
85
<210> 3
<211> 167
<212> PRT
<213> Escherichia coli
<400> 3
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro
35 40 45
Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp
165
<210> 4
<211> 167
<212> PRT
<213> Escherichia coli
<400> 4
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala
35 40 45
Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp
165
<210> 5
<211> 3990
<212> DNA
<213> Artificial Sequence
<220>
<223> ASCPF1-2NLS
<400> 5
atgacgcagt tcgaggggtt caccaacctc taccaggtca gcaagacgct gcggttcgag 60
ctgattccgc agggcaagac cctcaagcac atccaggagc agggctttat cgaggaggac 120
aaagcgcgga acgaccacta caaggagctc aagccgatca tcgaccggat ctacaagacc 180
tacgcggacc agtgcctgca gctcgtgcag ctcgactggg agaacctctc ggccgccatc 240
gactcctacc gcaaggagaa gacggaggag acacgcaacg cgctcatcga agaacaggct 300
acgtatcgca acgctatcca cgactacttc atcgggcgca cagataatct caccgacgcc 360
atcaacaagc gccacgccga aatctataag gggctgttta aggccgagct gttcaatggc 420
aaagtgctga agcaactggg caccgtcacg accacagagc atgagaacgc cctcctccgg 480
tcgttcgaca agtttacgac atactttagc ggcttttacg agaaccgcaa aaacgtgttc 540
agcgccgagg acattagcac cgccatcccg cataggatcg tgcaagacaa cttcccgaag 600
ttcaaggaga actgccacat cttcacccgc ctcatcacgg ccgtgccgtc cctccgcgag 660
cacttcgaga acgtgaagaa ggccatcggg atcttcgtgt ccacctcgat cgaagaggtg 720
ttctccttcc ccttctacaa tcagctgctg acgcagaccc agatcgacct ctacaaccag 780
ctcctcggcg gcatctcccg cgaggccggc accgagaaga tcaagggcct gaacgaggtg 840
ctcaatctcg ccatccagaa gaacgacgaa accgcgcaca tcattgcctc actcccacat 900
aggtttatcc ccctgtttaa gcagatcctc tccgaccgca acacgttgtc cttcatcctc 960
gaggagttca agagcgacga ggaggtcatc cagtccttct gcaagtacaa gaccctcctc 1020
cgcaacgaga atgtgctcga aaccgccgag gcgctgttca atgagctcaa tagcatcgat 1080
ctcacccaca tcttcatctc ccataagaag ctggaaacca tctccagcgc cctgtgcgac 1140
cactgggaca ccctccgcaa cgccctctac gagcggcgca tcagcgagct caccggcaag 1200
atcacgaagt cggcgaaaga gaaagtgcaa aggagcctca agcacgagga cattaacctg 1260
caggagatca tctccgccgc gggcaaggag ctgtccgagg ctttcaagca gaagacctcg 1320
gagatcctct ctcacgccca cgcggccctg gaccagccgc tgccgacgac cctgaaaaag 1380
caagaggaaa aggaaatcct caagtcccag ctggacagcc tcctggggct gtaccacctt 1440
ctcgactggt tcgccgtgga cgagtccaac gaggtcgatc cggagtttag cgcccgcctc 1500
accgggatca agcttgagat ggaacctagc ttgagcttct ataataaggc gcgcaattac 1560
gcgaccaaga agccgtattc cgtggagaag ttcaagctga acttccaaat gcccaccctg 1620
gccagcgggt gggacgttaa caaagagaag aacaacggag ccattctttt cgtgaaaaat 1680
gggttgtatt atttgggaat tatgccgaaa caaaaaggca ggtacaaggc gctcagcttc 1740
gagccaactg agaagacctc cgagggcttc gacaagatgt actacgatta ttttcctgac 1800
gctgcaaaga tgataccgaa gtgcagcact cagcttaagg cggtgacggc gcactttcag 1860
acccatacca cccccatcct cctctccaac aacttcatcg agccgctcga gatcaccaag 1920
gagatatacg atctgaataa tccagaaaag gaacccaaga agttccagac cgcctacgcc 1980
aagaagacgg gcgatcaaaa ggggtataga gaggcgctct gcaagtggat cgacttcacg 2040
cgcgatttcc tcagcaagta caccaagaca acctccatcg atctctcttc cctccgcccc 2100
tcttcccagt acaaggacct cggggagtac tacgccgaac tcaacccact cctgtatcac 2160
atctcgtttc agcgtatcgc ggaaaaggag atcatggacg ccgtcgaaac cggcaagttg 2220
tatcttttcc aaatctataa caaggacttc gcgaagggcc accacgggaa gccaaacctg 2280
cacaccctct actggacagg cctcttcagc ccggaaaatc tcgcgaagac gagcataaag 2340
ctgaacggcc aggcagaact cttctacagg ccgaagtcca ggatgaagcg catggctcat 2400
cgcctcggtg agaagatgct gaacaagaag ctgaaagatc aaaagacgcc aatccctgat 2460
acactgtatc aggagctgta cgattacgtg aaccaccgcc tctcacacga cctcagcgac 2520
gaggcccgcg cgctcctgcc aaacgtcatc acgaaggagg tcagccacga gatcataaag 2580
gatcggcggt ttacctctga caagttcttt ttccatgtcc ccatcacgct gaactaccag 2640
gccgcgaata gcccgtccaa gttcaaccag cgggtcaacg cgtatctcaa ggagcaccca 2700
gagacaccca taatcgggat tgaccggggg gagcggaacc tcatctacat caccgtcatc 2760
gactccaccg gaaagattct cgagcaacgg tcgctcaata ccatccagca gttcgactac 2820
cagaagaagc tcgacaaccg ggagaaggaa cgcgtcgccg cgaggcaggc ctggtccgta 2880
gtgggcacga tcaaagacct gaagcagggc tatctcagcc aggtcatcca tgagatagtg 2940
gatctcatga tccactacca agccgtcgtg gtcctcgaga atctcaattt cggattcaaa 3000
tccaagcgca caggcatcgc cgagaaggcg gtgtaccaac agttcgagaa aatgcttatc 3060
gacaagctca attgcctggt gctcaaggac tatccggcgg agaaggtcgg gggggtcctc 3120
aatccgtatc agctgaccga ccagtttacg tcatttgcga agatgggcac ccagagcggc 3180
ttccttttct atgtcccggc cccatatacc tcaaagattg atcccttgac cggattcgtg 3240
gacccgtttg tctggaagac catcaagaac catgagtcgc gtaagcattt cctggagggt 3300
ttcgacttcc tgcactatga tgtaaaaacc ggagacttca tcctgcattt caagatgaat 3360
cggaacctct ccttccagcg gggactccct ggcttcatgc ccgcttggga tatcgttttt 3420
gagaaaaatg aaacccaatt cgacgccaaa ggcacgcctt tcatcgcggg caagaggatt 3480
gtccctgtaa ttgagaacca tagattcacc gggcgttacc gtgacctgta ccccgcaaac 3540
gaactcatcg ccctcctgga ggagaaaggc atcgttttcc gcgacgggtc aaatatcctc 3600
cccaaactgc tcgagaacga tgatagccac gctattgaca cgatggtagc gctcatcaga 3660
tccgtgctgc aaatgagaaa ttcaaatgct gccactgggg aggattacat caactcccct 3720
gtgcgtgatc tcaatggcgt gtgcttcgat tctagatttc agaatcctga gtggccgatg 3780
gatgccgatg ctaacggcgc ataccacata gcattgaaag gacaactgtt gttgaaccat 3840
ctcaaggaga gcaaggacct taagctgcag aacggcatca gcaaccagga ttggcttgcc 3900
tatatccaag agctccgcaa ttccggcggc agcccaaaga agaagaggaa ggtgagcggc 3960
ggcagcccaa agaagaagcg caaggtctag 3990
<210> 6
<211> 3969
<212> DNA
<213> Artificial Sequence
<220>
<223> FNCPF1-2NLS
<400> 6
atgtccatct accaggagtt cgtcaataag tactcactct ctaagaccct gcggttcgag 60
ctgatcccgc agggcaagac actcgagaac atcaaggcgc gcggcctgat tctcgacgat 120
gagaagcggg ccaaggacta caagaaggcg aagcagatca ttgataagta ccaccagttc 180
ttcatcgagg agattctgtc cagcgtgtgc atctctgagg atctcctgca gaattactcc 240
gacgtctact tcaagctcaa gaagtctgac gatgacaacc tgcagaagga tttcaagtcc 300
gccaaggaca ccatcaagaa gcagatttct gagtacatca aggattccga gaagttcaag 360
aatctcttca accagaatct gattgatgcg aagaagggcc aggagtctga cctgatcctc 420
tggctgaagc agtccaagga caatggcatt gagctgttca aggccaacag cgatatcacc 480
gatattgacg aggcgctgga gatcattaag tcattcaagg gctggaccac atacttcaag 540
ggcttccatg agaaccggaa gaatgtgtac tcatctaacg acattccgac ctccatcatc 600
tacaggatcg tcgatgacaa tctgccaaag ttcctcgaga acaaggccaa gtacgagtcc 660
ctcaaggaca aggccccgga ggcgattaat tacgagcaga tcaagaagga tctggcggag 720
gagctgacct tcgatatcga ctacaagaca agcgaggtga accagagggt gttctccctc 780
gatgaggtgt tcgagatcgc caatttcaac aattacctga accagtccgg cattaccaag 840
ttcaatacaa tcattggcgg caagttcgtc aacggcgaga ataccaagcg caagggcatt 900
aacgagtaca tcaatctcta ctcccagcag atcaacgaca agaccctgaa gaagtacaag 960
atgtctgtgc tcttcaagca gatcctgtcc gatacagagt ccaagagctt cgtcattgat 1020
aagctcgagg acgacagcga cgtggtcacc acaatgcagt cattctacga gcagatcgcc 1080
gcgttcaaga ccgtggagga gaagagcatt aaggagacac tctcactcct gttcgatgac 1140
ctgaaggccc agaagctcga cctgagcaag atctacttca agaacgataa gagcctcaca 1200
gacctgtcac agcaggtgtt cgatgactac tcagtgattg gcaccgccgt cctcgagtac 1260
attacacagc agatcgcgcc aaagaacctc gataatcctt ctaagaagga gcaggagctg 1320
atcgccaaga aaaccgagaa ggcgaagtac ctctccctgg agacaattaa gctcgccctg 1380
gaggagttca ataagcacag ggatattgac aagcagtgcc gcttcgagga gatcctcgcg 1440
aacttcgccg cgatcccaat gattttcgat gagatcgccc agaacaagga caatctggcg 1500
cagatctcta ttaagtacca gaaccagggc aagaaggacc tcctgcaggc ctccgcagag 1560
gacgacgtga aggccatcaa ggatctcctg gaccagacca acaatctcct gcacaagctc 1620
aagatcttcc atatttcaca gtctgaggat aaggccaata tcctcgataa ggacgagcat 1680
ttctacctgg tgttcgagga gtgctacttc gagctggcga acattgtccc tctgtacaac 1740
aagattagga attacatcac acagaagccg tacagcgacg agaagttcaa gctcaacttc 1800
gagaattcaa ccctggccaa cggctgggat aagaataagg agcctgacaa cacagcgatc 1860
ctcttcatca aggacgacaa gtactacctg ggcgtgatga ataagaagaa caataagatc 1920
ttcgatgaca aggccattaa ggagaacaag ggcgagggct acaagaagat cgtgtacaag 1980
ctcctgcctg gcgccaataa gatgctcccg aaggtgttct tctccgcgaa gtccattaag 2040
ttctacaacc caagcgagga tatcctcagg atcaggaacc actctaccca tacaaagaac 2100
ggctcccctc agaagggcta cgagaagttc gagttcaata tcgaggattg ccggaagttc 2160
attgacttct acaagcagtc catcagcaag caccctgagt ggaaggattt cggcttccgc 2220
ttcagcgaca cccagcggta caactcaatc gatgagttct acagggaggt ggagaatcag 2280
ggctacaagc tcacattcga gaacatttca gagtcttaca tcgactccgt ggtcaatcag 2340
ggcaagctct acctgttcca gatctacaac aaggatttca gcgcctactc aaagggcagg 2400
ccgaacctcc ataccctgta ctggaaggcg ctcttcgatg agcgcaatct gcaggacgtg 2460
gtctacaagc tcaacggcga ggccgagctg ttctaccgca agcagtctat tccgaagaag 2520
atcacacacc cagcgaagga ggccatcgcg aacaagaata aggacaatcc gaagaaggag 2580
tccgtgttcg agtacgatct cattaaggac aagcggttca ccgaggataa gttcttcttc 2640
cattgcccaa tcacaattaa cttcaagtcc agcggcgcca acaagttcaa tgacgagatc 2700
aatctcctgc tcaaggagaa ggcgaacgat gtgcatatcc tctccattga ccgcggcgag 2760
cggcatctcg cctactacac cctggtcgat ggcaagggca atatcattaa gcaggatacc 2820
ttcaacatca ttggcaatga ccgcatgaaa accaactacc acgataagct cgccgcgatc 2880
gagaaggata gggacagcgc gcgcaaggac tggaagaaga tcaacaatat taaggagatg 2940
aaggagggct acctctcaca ggtggtccat gagattgcca agctcgtgat cgagtacaat 3000
gcgattgtgg tgttcgagga cctcaacttc ggcttcaagc ggggcaggtt caaggtggag 3060
aagcaggtct accagaagct ggagaagatg ctcatcgaga agctcaatta cctggtgttc 3120
aaggataacg agttcgacaa gaccggagga gtcctcaggg cataccagct gaccgcgcca 3180
ttcgagacat tcaagaagat gggcaagcag acaggcatca tctactacgt gccagccggc 3240
ttcacctcca agatctgccc tgtgacaggc ttcgtcaacc agctctaccc taagtacgag 3300
tccgtcagca agtcacagga gttcttcagc aagttcgata agatttgcta caatctggac 3360
aagggctact tcgagttctc attcgattac aagaacttcg gcgacaaggc cgcgaagggc 3420
aagtggacca ttgcgtcttt cggctcccgg ctcatcaact tcaggaatag cgataagaac 3480
cacaattggg acaccaggga ggtgtacccg acaaaggagc tggagaagct gctcaaggac 3540
tactcaatcg agtacggcca tggcgagtgc attaaggccg cgatctgcgg cgagagcgat 3600
aagaagttct tcgccaagct gacctcagtc ctcaatacaa tcctgcagat gaggaactct 3660
aagaccggca cagagctgga ctacctgatt tccccagtgg ccgatgtcaa cggcaatttc 3720
ttcgacagcc gccaggcgcc gaagaatatg ccacaggacg ccgacgccaa cggcgcatac 3780
cacatcggcc tcaagggcct gatgctgctc ggacgcatta agaacaatca ggagggcaag 3840
aagctcaacc tcgtgatcaa gaacgaggag tacttcgagt tcgtccaaaa ccggaacaat 3900
tccggcggca gcccaaagaa gaagaggaag gtgagcggcg gcagcccaaa gaagaagcgc 3960
aaggtctag 3969
<210> 7
<211> 3753
<212> DNA
<213> Artificial Sequence
<220>
<223> LBCPF1-2NLS
<400> 7
atgtcaaagc tcgagaaatt caccaactgt tattcgttga gcaaaacact gcggtttaaa 60
gcgattccag tcggcaagac tcaagagaat atagacaata agcggctgtt ggtggaagat 120
gaaaagcgcg cggaagacta caaaggggtg aagaagttgt tggacagata ctacctctct 180
tttatcaatg atgtcttgca ctcaatcaaa ttgaagaatc tgaacaacta catctccctc 240
ttcagaaaga aaacaaggac agaaaaggag aataaggaac ttgaaaattt ggagatcaat 300
ctgaggaaag agatcgcgaa agcctttaaa ggcaacgaag gatacaaaag tctgttcaag 360
aaggatataa ttgagacaat tttgccagag ttcctcgatg acaaggacga gattgcgctg 420
gtcaattcgt tcaacggatt cacaacagca ttcacaggct tctttgataa tcgggaaaat 480
atgttctctg aggaggcaaa gtccacttct attgcgttca ggtgtatcaa tgagaatctc 540
actaggtaca tttccaacat ggatatcttt gagaaggttg acgcaatttt tgacaagcac 600
gaagttcagg agattaagga gaagatcctc aattccgatt atgacgttga ggacttcttc 660
gaaggtgagt tttttaattt cgtgctcact caagagggta tcgacgtgta taatgcgatc 720
atcggtgggt tcgtgactga gtccggtgaa aagattaagg gattgaacga gtatatcaac 780
ctttacaacc aaaagacgaa acagaagctg ccaaagttca agcctcttta caaacaggtt 840
ctttcagacc gcgagtcact ctcgttctat ggggagggct acacttcgga tgaggaagtc 900
ctggaggtgt tcaggaatac tctcaataag aattcggaga ttttctcttc tataaaaaaa 960
ctggaaaagt tgtttaagaa ttttgacgaa tactctagcg ccggcatatt tgtgaaaaac 1020
ggcccggcca tatcaacgat aagtaaagat atcttcggcg aatggaacgt gatcagagac 1080
aaatggaacg cggagtatga cgatattcac ctgaagaaga aggctgtcgt aacggagaag 1140
tacgaggatg atcgcaggaa aagcttcaaa aagatcggaa gtttcagcct ggaacagttg 1200
caggagtatg ctgacgccga tcttagcgtc gtcgagaagt tgaaggagat aatcatccaa 1260
aaggtcgacg agatatataa agtctatgga tcaagtgaaa aactgttcga cgccgacttc 1320
gttttggaga agtccctgaa gaagaacgac gctgttgttg ccattatgaa ggatctgctc 1380
gacagcgtga agagtttcga gaactatatt aaggcttttt tcggggaggg gaaggagact 1440
aacagagatg agtccttcta cggagacttc gtcctcgcgt acgatatact ccttaaggta 1500
gaccacatct acgacgcaat cagaaattac gtgacacaaa agccgtacag caaggacaag 1560
ttcaaactct acttccagaa cccccagttc atgggcggct gggacaagga caaggaaacg 1620
gattacaggg ctacgatcct gaggtatggt tcaaaatact acttggcgat tatggacaag 1680
aagtacgcca agtgtctcca gaagattgac aaagacgatg tcaatggcaa ttatgagaag 1740
atcaactaca agctgcttcc gggtccgaac aagatgctcc caaaggtttt cttcagcaag 1800
aaatggatgg cctactataa cccaagcgag gacatccaga agatttataa gaacggtacg 1860
ttcaagaagg gcgacatgtt caatcttaac gactgtcaca agctgatcga cttcttcaaa 1920
gactcaatta gccggtaccc aaagtggtct aacgcctatg acttcaactt ttcggaaacc 1980
gagaagtaca aggatatagc cggattttat agagaggtgg aagagcaggg ctacaaggtg 2040
tcattcgagt ccgccagcaa gaaggaagtg gacaagctcg tggaagaggg taagctctac 2100
atgttccaga tttataataa agactttagc gataagagcc acgggacacc taatctccac 2160
acaatgtatt tcaagctgct cttcgacgag aataaccacg gccaaatcag gttgtcagga 2220
ggggctgaac tcttcatgcg gcgcgctagc cttaagaagg aggagcttgt agtccaccct 2280
gcgaatagtc caattgcgaa taagaacccg gacaatccta aaaagactac aacattgagc 2340
tacgacgtgt acaaggataa gaggttttcc gaggatcagt acgagctcca catcccgatt 2400
gcgatcaaca agtgcccaaa gaatattttc aagataaaca cagaggtgcg tgtactcctg 2460
aagcatgacg acaatcctta cgtcattggg attgatcggg gcgagaggaa cctcctctat 2520
attgtggtgg tggacgggaa ggggaacata gtcgaacagt actcccttaa cgaaataatt 2580
aacaatttca acggcatccg tatcaagacc gactaccatt cgttgctgga caagaaggag 2640
aaggagagat ttgaggcgcg gcaaaattgg acaagtatcg agaacatcaa ggaactcaaa 2700
gcaggttata tctctcaagt tgtgcataag atatgcgagc tggttgagaa gtatgacgca 2760
gtgatcgctc ttgaggacct caactcgggc tttaagaatt ctagagttaa agtggagaag 2820
caggtctatc aaaagttcga gaagatgctt atagataagc tcaactacat ggtcgataag 2880
aaatcgaacc catgtgccac cggcggcgca ctcaaaggtt accaaataac aaacaaattc 2940
gagtccttca aatcgatgag tactcagaat gggttcatat tttatatacc ggcgtggctt 3000
acgtctaaga tcgacccgtc aactggtttt gtcaacctgt tgaagacgaa atacacgtcc 3060
attgccgatt cgaaaaagtt catatctagt tttgatcgta ttatgtacgt cccagaggaa 3120
gatcttttcg agtttgctct cgactacaaa aacttttcgc ggaccgatgc ggattacatt 3180
aaaaaatgga aactctattc gtacggcaac agaatcagga tttttcgcaa ccctaagaag 3240
aataacgtct ttgattggga ggaagtttgc ttgactagcg cgtacaagga gctctttaat 3300
aagtatggca ttaactacca acagggtgat atcagagcac tgctttgcga acaatctgac 3360
aaggctttct actcatcctt catggctttg atgagcctga tgctccagat gagaaattca 3420
attacaggca gaaccgacgt ggatttcttg atctccccgg ttaaaaattc tgatggcatc 3480
ttttacgata gcaggaacta tgaagcgcaa gagaatgcga ttctgccaaa aaatgcagac 3540
gccaacggtg cctataacat cgccaggaaa gtcctgtggg cgatcggcca gttcaaaaag 3600
gccgaagacg aaaaattgga caaggtcaaa atcgctatca gcaacaaaga gtggctggag 3660
tatgctcaga catccgtaaa gcattccggc ggcagcccaa agaagaagag gaaggtgagc 3720
ggcggcagcc caaagaagaa gcgcaaggtc tag 3753
<210> 8
<211> 4995
<212> DNA
<213> Artificial Sequence
<220>
<223> dFNCPF1-PBE-2NLS
<400> 8
atgccaaaga agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc 60
ctgcggcgga gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc 120
aaggagacgt gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac 180
accagccaaa acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag 240
aggtacttct gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc 300
ggcgagtgct ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc 360
atctacatcg ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac 420
ctgatctcca gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg 480
aggaacttcg tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc 540
tgggtgcgcc tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc 600
aacatcctga ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc 660
cactaccaga ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag 720
acgccaggca cctccgagag cgctacgcct gaacttaagt ccatctacca ggagttcgtc 780
aataagtact cactctctaa gaccctgcgg ttcgagctga tcccgcaggg caagacactc 840
gagaacatca aggcgcgcgg cctgattctc gacgatgaga agcgggccaa ggactacaag 900
aaggcgaagc agatcattga taagtaccac cagttcttca tcgaggagat tctgtccagc 960
gtgtgcatct ctgaggatct cctgcagaat tactccgacg tctacttcaa gctcaagaag 1020
tctgacgatg acaacctgca gaaggatttc aagtccgcca aggacaccat caagaagcag 1080
atttctgagt acatcaagga ttccgagaag ttcaagaatc tcttcaacca gaatctgatt 1140
gatgcgaaga agggccagga gtctgacctg atcctctggc tgaagcagtc caaggacaat 1200
ggcattgagc tgttcaaggc caacagcgat atcaccgata ttgacgaggc gctggagatc 1260
attaagtcat tcaagggctg gaccacatac ttcaagggct tccatgagaa ccggaagaat 1320
gtgtactcat ctaacgacat tccgacctcc atcatctaca ggatcgtcga tgacaatctg 1380
ccaaagttcc tcgagaacaa ggccaagtac gagtccctca aggacaaggc cccggaggcg 1440
attaattacg agcagatcaa gaaggatctg gcggaggagc tgaccttcga tatcgactac 1500
aagacaagcg aggtgaacca gagggtgttc tccctcgatg aggtgttcga gatcgccaat 1560
ttcaacaatt acctgaacca gtccggcatt accaagttca atacaatcat tggcggcaag 1620
ttcgtcaacg gcgagaatac caagcgcaag ggcattaacg agtacatcaa tctctactcc 1680
cagcagatca acgacaagac cctgaagaag tacaagatgt ctgtgctctt caagcagatc 1740
ctgtccgata cagagtccaa gagcttcgtc attgataagc tcgaggacga cagcgacgtg 1800
gtcaccacaa tgcagtcatt ctacgagcag atcgccgcgt tcaagaccgt ggaggagaag 1860
agcattaagg agacactctc actcctgttc gatgacctga aggcccagaa gctcgacctg 1920
agcaagatct acttcaagaa cgataagagc ctcacagacc tgtcacagca ggtgttcgat 1980
gactactcag tgattggcac cgccgtcctc gagtacatta cacagcagat cgcgccaaag 2040
aacctcgata atccttctaa gaaggagcag gagctgatcg ccaagaaaac cgagaaggcg 2100
aagtacctct ccctggagac aattaagctc gccctggagg agttcaataa gcacagggat 2160
attgacaagc agtgccgctt cgaggagatc ctcgcgaact tcgccgcgat cccaatgatt 2220
ttcgatgaga tcgcccagaa caaggacaat ctggcgcaga tctctattaa gtaccagaac 2280
cagggcaaga aggacctcct gcaggcctcc gcagaggacg acgtgaaggc catcaaggat 2340
ctcctggacc agaccaacaa tctcctgcac aagctcaaga tcttccatat ttcacagtct 2400
gaggataagg ccaatatcct cgataaggac gagcatttct acctggtgtt cgaggagtgc 2460
tacttcgagc tggcgaacat tgtccctctg tacaacaaga ttaggaatta catcacacag 2520
aagccgtaca gcgacgagaa gttcaagctc aacttcgaga attcaaccct ggccaacggc 2580
tgggataaga ataaggagcc tgacaacaca gcgatcctct tcatcaagga cgacaagtac 2640
tacctgggcg tgatgaataa gaagaacaat aagatcttcg atgacaaggc cattaaggag 2700
aacaagggcg agggctacaa gaagatcgtg tacaagctcc tgcctggcgc caataagatg 2760
ctcccgaagg tgttcttctc cgcgaagtcc attaagttct acaacccaag cgaggatatc 2820
ctcaggatca ggaaccactc tacccataca aagaacggct cccctcagaa gggctacgag 2880
aagttcgagt tcaatatcga ggattgccgg aagttcattg acttctacaa gcagtccatc 2940
agcaagcacc ctgagtggaa ggatttcggc ttccgcttca gcgacaccca gcggtacaac 3000
tcaatcgatg agttctacag ggaggtggag aatcagggct acaagctcac attcgagaac 3060
atttcagagt cttacatcga ctccgtggtc aatcagggca agctctacct gttccagatc 3120
tacaacaagg atttcagcgc ctactcaaag ggcaggccga acctccatac cctgtactgg 3180
aaggcgctct tcgatgagcg caatctgcag gacgtggtct acaagctcaa cggcgaggcc 3240
gagctgttct accgcaagca gtctattccg aagaagatca cacacccagc gaaggaggcc 3300
atcgcgaaca agaataagga caatccgaag aaggagtccg tgttcgagta cgatctcatt 3360
aaggacaagc ggttcaccga ggataagttc ttcttccatt gcccaatcac aattaacttc 3420
aagtccagcg gcgccaacaa gttcaatgac gagatcaatc tcctgctcaa ggagaaggcg 3480
aacgatgtgc atatcctctc cattgcccgc ggcgagcggc atctcgccta ctacaccctg 3540
gtcgatggca agggcaatat cattaagcag gataccttca acatcattgg caatgaccgc 3600
atgaaaacca actaccacga taagctcgcc gcgatcgaga aggataggga cagcgcgcgc 3660
aaggactgga agaagatcaa caatattaag gagatgaagg agggctacct ctcacaggtg 3720
gtccatgaga ttgccaagct cgtgatcgag tacaatgcga ttgtggtgtt cgaggacctc 3780
aacttcggct tcaagcgggg caggttcaag gtggagaagc aggtctacca gaagctggag 3840
aagatgctca tcgagaagct caattacctg gtgttcaagg ataacgagtt cgacaagacc 3900
ggaggagtcc tcagggcata ccagctgacc gcgccattcg agacattcaa gaagatgggc 3960
aagcagacag gcatcatcta ctacgtgcca gccggcttca cctccaagat ctgccctgtg 4020
acaggcttcg tcaaccagct ctaccctaag tacgagtccg tcagcaagtc acaggagttc 4080
ttcagcaagt tcgataagat ttgctacaat ctggacaagg gctacttcga gttctcattc 4140
gattacaaga acttcggcga caaggccgcg aagggcaagt ggaccattgc gtctttcggc 4200
tcccggctca tcaacttcag gaatagcgat aagaaccaca attgggacac cagggaggtg 4260
tacccgacaa aggagctgga gaagctgctc aaggactact caatcgagta cggccatggc 4320
gagtgcatta aggccgcgat ctgcggcgag agcgataaga agttcttcgc caagctgacc 4380
tcagtcctca atacaatcct gcagatgagg aactctaaga ccggcacaga gctggactac 4440
ctgatttccc cagtggccga tgtcaacggc aatttcttcg acagccgcca ggcgccgaag 4500
aatatgccac aggacgccga cgccaacggc gcataccaca tcggcctcaa gggcctgatg 4560
ctgctcggac gcattaagaa caatcaggag ggcaagaagc tcaacctcgt gatcaagaac 4620
gaggagtact tcgagttcgt ccaaaaccgg aacaatacgc gtgactccgg cggcagcacc 4680
aacctgtccg acatcatcga gaaggagacg ggcaagcaac tcgtgatcca ggagagcatc 4740
ctcatgctgc cagaggaggt ggaggaggtc atcggcaaca agccagagtc cgacatcctg 4800
gtgcacaccg cctacgacga gtccaccgac gagaacgtca tgctcctgac cagcgacgcc 4860
ccagagtaca agccatgggc cctcgtcatc caggacagca acggggagaa caagatcaag 4920
atgctgtcgg gggggagccc aaagaagaag cggaaggtga gcggcggcag cccaaagaag 4980
aagcgcaagg tctag 4995
<210> 9
<211> 4779
<212> DNA
<213> Artificial Sequence
<220>
<223> dLBCPF1-PBE-2NLS
<400> 9
atgccaaaga agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc 60
ctgcggcgga gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc 120
aaggagacgt gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac 180
accagccaaa acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag 240
aggtacttct gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc 300
ggcgagtgct ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc 360
atctacatcg ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac 420
ctgatctcca gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg 480
aggaacttcg tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc 540
tgggtgcgcc tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc 600
aacatcctga ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc 660
cactaccaga ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag 720
acgccaggca cctccgagag cgctacgcct gaacttaagt caaagctcga gaaattcacc 780
aactgttatt cgttgagcaa aacactgcgg tttaaagcga ttccagtcgg caagactcaa 840
gagaatatag acaataagcg gctgttggtg gaagatgaaa agcgcgcgga agactacaaa 900
ggggtgaaga agttgttgga cagatactac ctctctttta tcaatgatgt cttgcactca 960
atcaaattga agaatctgaa caactacatc tccctcttca gaaagaaaac aaggacagaa 1020
aaggagaata aggaacttga aaatttggag atcaatctga ggaaagagat cgcgaaagcc 1080
tttaaaggca acgaaggata caaaagtctg ttcaagaagg atataattga gacaattttg 1140
ccagagttcc tcgatgacaa ggacgagatt gcgctggtca attcgttcaa cggattcaca 1200
acagcattca caggcttctt tgataatcgg gaaaatatgt tctctgagga ggcaaagtcc 1260
acttctattg cgttcaggtg tatcaatgag aatctcacta ggtacatttc caacatggat 1320
atctttgaga aggttgacgc aatttttgac aagcacgaag ttcaggagat taaggagaag 1380
atcctcaatt ccgattatga cgttgaggac ttcttcgaag gtgagttttt taatttcgtg 1440
ctcactcaag agggtatcga cgtgtataat gcgatcatcg gtgggttcgt gactgagtcc 1500
ggtgaaaaga ttaagggatt gaacgagtat atcaaccttt acaaccaaaa gacgaaacag 1560
aagctgccaa agttcaagcc tctttacaaa caggttcttt cagaccgcga gtcactctcg 1620
ttctatgggg agggctacac ttcggatgag gaagtcctgg aggtgttcag gaatactctc 1680
aataagaatt cggagatttt ctcttctata aaaaaactgg aaaagttgtt taagaatttt 1740
gacgaatact ctagcgccgg catatttgtg aaaaacggcc cggccatatc aacgataagt 1800
aaagatatct tcggcgaatg gaacgtgatc agagacaaat ggaacgcgga gtatgacgat 1860
attcacctga agaagaaggc tgtcgtaacg gagaagtacg aggatgatcg caggaaaagc 1920
ttcaaaaaga tcggaagttt cagcctggaa cagttgcagg agtatgctga cgccgatctt 1980
agcgtcgtcg agaagttgaa ggagataatc atccaaaagg tcgacgagat atataaagtc 2040
tatggatcaa gtgaaaaact gttcgacgcc gacttcgttt tggagaagtc cctgaagaag 2100
aacgacgctg ttgttgccat tatgaaggat ctgctcgaca gcgtgaagag tttcgagaac 2160
tatattaagg cttttttcgg ggaggggaag gagactaaca gagatgagtc cttctacgga 2220
gacttcgtcc tcgcgtacga tatactcctt aaggtagacc acatctacga cgcaatcaga 2280
aattacgtga cacaaaagcc gtacagcaag gacaagttca aactctactt ccagaacccc 2340
cagttcatgg gcggctggga caaggacaag gaaacggatt acagggctac gatcctgagg 2400
tatggttcaa aatactactt ggcgattatg gacaagaagt acgccaagtg tctccagaag 2460
attgacaaag acgatgtcaa tggcaattat gagaagatca actacaagct gcttccgggt 2520
ccgaacaaga tgctcccaaa ggttttcttc agcaagaaat ggatggccta ctataaccca 2580
agcgaggaca tccagaagat ttataagaac ggtacgttca agaagggcga catgttcaat 2640
cttaacgact gtcacaagct gatcgacttc ttcaaagact caattagccg gtacccaaag 2700
tggtctaacg cctatgactt caacttttcg gaaaccgaga agtacaagga tatagccgga 2760
ttttatagag aggtggaaga gcagggctac aaggtgtcat tcgagtccgc cagcaagaag 2820
gaagtggaca agctcgtgga agagggtaag ctctacatgt tccagattta taataaagac 2880
tttagcgata agagccacgg gacacctaat ctccacacaa tgtatttcaa gctgctcttc 2940
gacgagaata accacggcca aatcaggttg tcaggagggg ctgaactctt catgcggcgc 3000
gctagcctta agaaggagga gcttgtagtc caccctgcga atagtccaat tgcgaataag 3060
aacccggaca atcctaaaaa gactacaaca ttgagctacg acgtgtacaa ggataagagg 3120
ttttccgagg atcagtacga gctccacatc ccgattgcga tcaacaagtg cccaaagaat 3180
attttcaaga taaacacaga ggtgcgtgta ctcctgaagc atgacgacaa tccttacgtc 3240
attgggattg ctcggggcga gaggaacctc ctctatattg tggtggtgga cgggaagggg 3300
aacatagtcg aacagtactc ccttaacgaa ataattaaca atttcaacgg catccgtatc 3360
aagaccgact accattcgtt gctggacaag aaggagaagg agagatttga ggcgcggcaa 3420
aattggacaa gtatcgagaa catcaaggaa ctcaaagcag gttatatctc tcaagttgtg 3480
cataagatat gcgagctggt tgagaagtat gacgcagtga tcgctcttga ggacctcaac 3540
tcgggcttta agaattctag agttaaagtg gagaagcagg tctatcaaaa gttcgagaag 3600
atgcttatag ataagctcaa ctacatggtc gataagaaat cgaacccatg tgccaccggc 3660
ggcgcactca aaggttacca aataacaaac aaattcgagt ccttcaaatc gatgagtact 3720
cagaatgggt tcatatttta tataccggcg tggcttacgt ctaagatcga cccgtcaact 3780
ggttttgtca acctgttgaa gacgaaatac acgtccattg ccgattcgaa aaagttcata 3840
tctagttttg atcgtattat gtacgtccca gaggaagatc ttttcgagtt tgctctcgac 3900
tacaaaaact tttcgcggac cgatgcggat tacattaaaa aatggaaact ctattcgtac 3960
ggcaacagaa tcaggatttt tcgcaaccct aagaagaata acgtctttga ttgggaggaa 4020
gtttgcttga ctagcgcgta caaggagctc tttaataagt atggcattaa ctaccaacag 4080
ggtgatatca gagcactgct ttgcgaacaa tctgacaagg ctttctactc atccttcatg 4140
gctttgatga gcctgatgct ccagatgaga aattcaatta caggcagaac cgacgtggat 4200
ttcttgatct ccccggttaa aaattctgat ggcatctttt acgatagcag gaactatgaa 4260
gcgcaagaga atgcgattct gccaaaaaat gcagacgcca acggtgccta taacatcgcc 4320
aggaaagtcc tgtgggcgat cggccagttc aaaaaggccg aagacgaaaa attggacaag 4380
gtcaaaatcg ctatcagcaa caaagagtgg ctggagtatg ctcagacatc cgtaaagcat 4440
acgcgtgact ccggcggcag caccaacctg tccgacatca tcgagaagga gacgggcaag 4500
caactcgtga tccaggagag catcctcatg ctgccagagg aggtggagga ggtcatcggc 4560
aacaagccag agtccgacat cctggtgcac accgcctacg acgagtccac cgacgagaac 4620
gtcatgctcc tgaccagcga cgccccagag tacaagccat gggccctcgt catccaggac 4680
agcaacgggg agaacaagat caagatgctg tcggggggga gcccaaagaa gaagcggaag 4740
gtgagcggcg gcagcccaaa gaagaagcgc aaggtctag 4779
<210> 10
<211> 6305
<212> DNA
<213> Artificial Sequence
<220>
<223> BDUBI10-dLBCPF1-PBE-2NLS
<400> 10
ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60
agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120
tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180
tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240
gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300
ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360
gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420
agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480
taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540
aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600
tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 660
cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 720
ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 780
gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 840
caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900
ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960
tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020
taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080
atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140
cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200
ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 1260
gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1320
cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1380
acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1440
ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1500
tctgcaggtc gaagcttgaa gcaaacatgc caaagaagaa gaggaaggtt tcatcggaga 1560
ccggccctgt tgctgttgac cccaccctgc ggcggagaat cgagccacac gagttcgagg 1620
tgttcttcga cccaagggag ctccgcaagg agacgtgcct cctgtacgag atcaactggg 1680
gcggcaggca ctccatctgg aggcacacca gccaaaacac caacaagcac gtggaggtca 1740
acttcatcga gaagttcacc accgagaggt acttctgccc aaacacccgc tgctccatca 1800
cctggttcct gtcctggagc ccatgcggcg agtgctccag ggccatcacc gagttcctca 1860
gccgctaccc acacgtcacc ctgttcatct acatcgccag gctctaccac cacgccgacc 1920
caaggaacag gcagggcctc cgcgacctga tctccagcgg cgtgaccatc caaatcatga 1980
ccgagcagga gtccggctac tgctggagga acttcgtcaa ctactcccca agcaacgagg 2040
cccactggcc aaggtaccca cacctctggg tgcgcctcta cgtgctcgag ctgtactgca 2100
tcatcctcgg cctgccacca tgcctcaaca tcctgaggcg caagcaacca cagctgacct 2160
tcttcaccat cgccctccaa agctgccact accagaggct cccaccacac atcctgtggg 2220
ctaccggcct caagtccggc agcgagacgc caggcacctc cgagagcgct acgcctgaac 2280
ttaagtcaaa gctcgagaaa ttcaccaact gttattcgtt gagcaaaaca ctgcggttta 2340
aagcgattcc agtcggcaag actcaagaga atatagacaa taagcggctg ttggtggaag 2400
atgaaaagcg cgcggaagac tacaaagggg tgaagaagtt gttggacaga tactacctct 2460
cttttatcaa tgatgtcttg cactcaatca aattgaagaa tctgaacaac tacatctccc 2520
tcttcagaaa gaaaacaagg acagaaaagg agaataagga acttgaaaat ttggagatca 2580
atctgaggaa agagatcgcg aaagccttta aaggcaacga aggatacaaa agtctgttca 2640
agaaggatat aattgagaca attttgccag agttcctcga tgacaaggac gagattgcgc 2700
tggtcaattc gttcaacgga ttcacaacag cattcacagg cttctttgat aatcgggaaa 2760
atatgttctc tgaggaggca aagtccactt ctattgcgtt caggtgtatc aatgagaatc 2820
tcactaggta catttccaac atggatatct ttgagaaggt tgacgcaatt tttgacaagc 2880
acgaagttca ggagattaag gagaagatcc tcaattccga ttatgacgtt gaggacttct 2940
tcgaaggtga gttttttaat ttcgtgctca ctcaagaggg tatcgacgtg tataatgcga 3000
tcatcggtgg gttcgtgact gagtccggtg aaaagattaa gggattgaac gagtatatca 3060
acctttacaa ccaaaagacg aaacagaagc tgccaaagtt caagcctctt tacaaacagg 3120
ttctttcaga ccgcgagtca ctctcgttct atggggaggg ctacacttcg gatgaggaag 3180
tcctggaggt gttcaggaat actctcaata agaattcgga gattttctct tctataaaaa 3240
aactggaaaa gttgtttaag aattttgacg aatactctag cgccggcata tttgtgaaaa 3300
acggcccggc catatcaacg ataagtaaag atatcttcgg cgaatggaac gtgatcagag 3360
acaaatggaa cgcggagtat gacgatattc acctgaagaa gaaggctgtc gtaacggaga 3420
agtacgagga tgatcgcagg aaaagcttca aaaagatcgg aagtttcagc ctggaacagt 3480
tgcaggagta tgctgacgcc gatcttagcg tcgtcgagaa gttgaaggag ataatcatcc 3540
aaaaggtcga cgagatatat aaagtctatg gatcaagtga aaaactgttc gacgccgact 3600
tcgttttgga gaagtccctg aagaagaacg acgctgttgt tgccattatg aaggatctgc 3660
tcgacagcgt gaagagtttc gagaactata ttaaggcttt tttcggggag gggaaggaga 3720
ctaacagaga tgagtccttc tacggagact tcgtcctcgc gtacgatata ctccttaagg 3780
tagaccacat ctacgacgca atcagaaatt acgtgacaca aaagccgtac agcaaggaca 3840
agttcaaact ctacttccag aacccccagt tcatgggcgg ctgggacaag gacaaggaaa 3900
cggattacag ggctacgatc ctgaggtatg gttcaaaata ctacttggcg attatggaca 3960
agaagtacgc caagtgtctc cagaagattg acaaagacga tgtcaatggc aattatgaga 4020
agatcaacta caagctgctt ccgggtccga acaagatgct cccaaaggtt ttcttcagca 4080
agaaatggat ggcctactat aacccaagcg aggacatcca gaagatttat aagaacggta 4140
cgttcaagaa gggcgacatg ttcaatctta acgactgtca caagctgatc gacttcttca 4200
aagactcaat tagccggtac ccaaagtggt ctaacgccta tgacttcaac ttttcggaaa 4260
ccgagaagta caaggatata gccggatttt atagagaggt ggaagagcag ggctacaagg 4320
tgtcattcga gtccgccagc aagaaggaag tggacaagct cgtggaagag ggtaagctct 4380
acatgttcca gatttataat aaagacttta gcgataagag ccacgggaca cctaatctcc 4440
acacaatgta tttcaagctg ctcttcgacg agaataacca cggccaaatc aggttgtcag 4500
gaggggctga actcttcatg cggcgcgcta gccttaagaa ggaggagctt gtagtccacc 4560
ctgcgaatag tccaattgcg aataagaacc cggacaatcc taaaaagact acaacattga 4620
gctacgacgt gtacaaggat aagaggtttt ccgaggatca gtacgagctc cacatcccga 4680
ttgcgatcaa caagtgccca aagaatattt tcaagataaa cacagaggtg cgtgtactcc 4740
tgaagcatga cgacaatcct tacgtcattg ggattgctcg gggcgagagg aacctcctct 4800
atattgtggt ggtggacggg aaggggaaca tagtcgaaca gtactccctt aacgaaataa 4860
ttaacaattt caacggcatc cgtatcaaga ccgactacca ttcgttgctg gacaagaagg 4920
agaaggagag atttgaggcg cggcaaaatt ggacaagtat cgagaacatc aaggaactca 4980
aagcaggtta tatctctcaa gttgtgcata agatatgcga gctggttgag aagtatgacg 5040
cagtgatcgc tcttgaggac ctcaactcgg gctttaagaa ttctagagtt aaagtggaga 5100
agcaggtcta tcaaaagttc gagaagatgc ttatagataa gctcaactac atggtcgata 5160
agaaatcgaa cccatgtgcc accggcggcg cactcaaagg ttaccaaata acaaacaaat 5220
tcgagtcctt caaatcgatg agtactcaga atgggttcat attttatata ccggcgtggc 5280
ttacgtctaa gatcgacccg tcaactggtt ttgtcaacct gttgaagacg aaatacacgt 5340
ccattgccga ttcgaaaaag ttcatatcta gttttgatcg tattatgtac gtcccagagg 5400
aagatctttt cgagtttgct ctcgactaca aaaacttttc gcggaccgat gcggattaca 5460
ttaaaaaatg gaaactctat tcgtacggca acagaatcag gatttttcgc aaccctaaga 5520
agaataacgt ctttgattgg gaggaagttt gcttgactag cgcgtacaag gagctcttta 5580
ataagtatgg cattaactac caacagggtg atatcagagc actgctttgc gaacaatctg 5640
acaaggcttt ctactcatcc ttcatggctt tgatgagcct gatgctccag atgagaaatt 5700
caattacagg cagaaccgac gtggatttct tgatctcccc ggttaaaaat tctgatggca 5760
tcttttacga tagcaggaac tatgaagcgc aagagaatgc gattctgcca aaaaatgcag 5820
acgccaacgg tgcctataac atcgccagga aagtcctgtg ggcgatcggc cagttcaaaa 5880
aggccgaaga cgaaaaattg gacaaggtca aaatcgctat cagcaacaaa gagtggctgg 5940
agtatgctca gacatccgta aagcatacgc gtgactccgg cggcagcacc aacctgtccg 6000
acatcatcga gaaggagacg ggcaagcaac tcgtgatcca ggagagcatc ctcatgctgc 6060
cagaggaggt ggaggaggtc atcggcaaca agccagagtc cgacatcctg gtgcacaccg 6120
cctacgacga gtccaccgac gagaacgtca tgctcctgac cagcgacgcc ccagagtaca 6180
agccatgggc cctcgtcatc caggacagca acggggagaa caagatcaag atgctgtcgg 6240
gggggagccc aaagaagaag cggaaggtga gcggcggcag cccaaagaag aagcgcaagg 6300
tctag 6305
<210> 11
<211> 5196
<212> DNA
<213> Artificial Sequence
<220>
<223> dFNCPF1-ABE7.10-2NLS
<400> 11
atgccaaaaa agaagagaaa ggtttcaggc ggctcctccg aggtggagtt ctctcacgag 60
tattggatga ggcacgctct tacacttgct aagagagctt gggacgaaag agaagtgcca 120
gttggcgccg ttcttgtgca taataatagg gtgatcggcg agggttggaa tagaccaatt 180
ggaaggcatg atccaacagc tcacgcagag attatggctc tcagacaagg cggcctcgtt 240
atgcagaact acaggctcat tgacgctaca ctctacgtga cactcgaacc ttgcgttatg 300
tgcgccggag ctatgattca ttctaggatt ggcagggtcg tgtttggagc tagggacgct 360
aaaacaggag ccgccggatc tcttatggac gtgttgcatc atccaggcat gaaccatagg 420
gtggagatta cagagggcat tcttgcagac gagtgcgctg ctcttctttc cgatttcttc 480
aggatgagaa ggcaggagat taaggcccag aagaaggctc agtcttctac agatagcgga 540
ggatcttccg gaggatctag cggctccgag acaccaggaa catccgaaag cgctacacca 600
gaatctagcg gaggctcttc cggaggatct tctgaagtgg agttctccca cgagtattgg 660
atgaggcacg ctcttacact tgctaaaagg gctagggacg aaagggaagt tccagttgga 720
gctgttctcg tgctcaataa cagggtgatt ggcgagggtt ggaatagagc cattggactc 780
catgatccaa cagctcacgc agagattatg gctcttagac aaggcggcct cgttatgcag 840
aattacagac tcatcgacgc cacactctac gttaccttcg aaccttgcgt tatgtgcgcc 900
ggagctatga tccattctag gattggcagg gtcgtgttcg gcgttagaaa cgctaagaca 960
ggagctgcag gctctcttat ggacgttctt cattacccag gcatgaatca tagagtggag 1020
atcacagaag gcattcttgc agacgagtgc gcagctctcc tttgctattt cttcaggatg 1080
ccgaggcaag ttttcaacgc tcagaagaag gcccagtctt ctacagattc cggcggatct 1140
tctggaggat ctagcggctc cgagacacca ggaacatccg aatccgctac accagagtct 1200
tctggaggat ctagcggagg atctcttaag tccatctacc aggagttcgt caataagtac 1260
tcactctcta agaccctgcg gttcgagctg atcccgcagg gcaagacact cgagaacatc 1320
aaggcgcgcg gcctgattct cgacgatgag aagcgggcca aggactacaa gaaggcgaag 1380
cagatcattg ataagtacca ccagttcttc atcgaggaga ttctgtccag cgtgtgcatc 1440
tctgaggatc tcctgcagaa ttactccgac gtctacttca agctcaagaa gtctgacgat 1500
gacaacctgc agaaggattt caagtccgcc aaggacacca tcaagaagca gatttctgag 1560
tacatcaagg attccgagaa gttcaagaat ctcttcaacc agaatctgat tgatgcgaag 1620
aagggccagg agtctgacct gatcctctgg ctgaagcagt ccaaggacaa tggcattgag 1680
ctgttcaagg ccaacagcga tatcaccgat attgacgagg cgctggagat cattaagtca 1740
ttcaagggct ggaccacata cttcaagggc ttccatgaga accggaagaa tgtgtactca 1800
tctaacgaca ttccgacctc catcatctac aggatcgtcg atgacaatct gccaaagttc 1860
ctcgagaaca aggccaagta cgagtccctc aaggacaagg ccccggaggc gattaattac 1920
gagcagatca agaaggatct ggcggaggag ctgaccttcg atatcgacta caagacaagc 1980
gaggtgaacc agagggtgtt ctccctcgat gaggtgttcg agatcgccaa tttcaacaat 2040
tacctgaacc agtccggcat taccaagttc aatacaatca ttggcggcaa gttcgtcaac 2100
ggcgagaata ccaagcgcaa gggcattaac gagtacatca atctctactc ccagcagatc 2160
aacgacaaga ccctgaagaa gtacaagatg tctgtgctct tcaagcagat cctgtccgat 2220
acagagtcca agagcttcgt cattgataag ctcgaggacg acagcgacgt ggtcaccaca 2280
atgcagtcat tctacgagca gatcgccgcg ttcaagaccg tggaggagaa gagcattaag 2340
gagacactct cactcctgtt cgatgacctg aaggcccaga agctcgacct gagcaagatc 2400
tacttcaaga acgataagag cctcacagac ctgtcacagc aggtgttcga tgactactca 2460
gtgattggca ccgccgtcct cgagtacatt acacagcaga tcgcgccaaa gaacctcgat 2520
aatccttcta agaaggagca ggagctgatc gccaagaaaa ccgagaaggc gaagtacctc 2580
tccctggaga caattaagct cgccctggag gagttcaata agcacaggga tattgacaag 2640
cagtgccgct tcgaggagat cctcgcgaac ttcgccgcga tcccaatgat tttcgatgag 2700
atcgcccaga acaaggacaa tctggcgcag atctctatta agtaccagaa ccagggcaag 2760
aaggacctcc tgcaggcctc cgcagaggac gacgtgaagg ccatcaagga tctcctggac 2820
cagaccaaca atctcctgca caagctcaag atcttccata tttcacagtc tgaggataag 2880
gccaatatcc tcgataagga cgagcatttc tacctggtgt tcgaggagtg ctacttcgag 2940
ctggcgaaca ttgtccctct gtacaacaag attaggaatt acatcacaca gaagccgtac 3000
agcgacgaga agttcaagct caacttcgag aattcaaccc tggccaacgg ctgggataag 3060
aataaggagc ctgacaacac agcgatcctc ttcatcaagg acgacaagta ctacctgggc 3120
gtgatgaata agaagaacaa taagatcttc gatgacaagg ccattaagga gaacaagggc 3180
gagggctaca agaagatcgt gtacaagctc ctgcctggcg ccaataagat gctcccgaag 3240
gtgttcttct ccgcgaagtc cattaagttc tacaacccaa gcgaggatat cctcaggatc 3300
aggaaccact ctacccatac aaagaacggc tcccctcaga agggctacga gaagttcgag 3360
ttcaatatcg aggattgccg gaagttcatt gacttctaca agcagtccat cagcaagcac 3420
cctgagtgga aggatttcgg cttccgcttc agcgacaccc agcggtacaa ctcaatcgat 3480
gagttctaca gggaggtgga gaatcagggc tacaagctca cattcgagaa catttcagag 3540
tcttacatcg actccgtggt caatcagggc aagctctacc tgttccagat ctacaacaag 3600
gatttcagcg cctactcaaa gggcaggccg aacctccata ccctgtactg gaaggcgctc 3660
ttcgatgagc gcaatctgca ggacgtggtc tacaagctca acggcgaggc cgagctgttc 3720
taccgcaagc agtctattcc gaagaagatc acacacccag cgaaggaggc catcgcgaac 3780
aagaataagg acaatccgaa gaaggagtcc gtgttcgagt acgatctcat taaggacaag 3840
cggttcaccg aggataagtt cttcttccat tgcccaatca caattaactt caagtccagc 3900
ggcgccaaca agttcaatga cgagatcaat ctcctgctca aggagaaggc gaacgatgtg 3960
catatcctct ccattgcccg cggcgagcgg catctcgcct actacaccct ggtcgatggc 4020
aagggcaata tcattaagca ggataccttc aacatcattg gcaatgaccg catgaaaacc 4080
aactaccacg ataagctcgc cgcgatcgag aaggataggg acagcgcgcg caaggactgg 4140
aagaagatca acaatattaa ggagatgaag gagggctacc tctcacaggt ggtccatgag 4200
attgccaagc tcgtgatcga gtacaatgcg attgtggtgt tcgaggacct caacttcggc 4260
ttcaagcggg gcaggttcaa ggtggagaag caggtctacc agaagctgga gaagatgctc 4320
atcgagaagc tcaattacct ggtgttcaag gataacgagt tcgacaagac cggaggagtc 4380
ctcagggcat accagctgac cgcgccattc gagacattca agaagatggg caagcagaca 4440
ggcatcatct actacgtgcc agccggcttc acctccaaga tctgccctgt gacaggcttc 4500
gtcaaccagc tctaccctaa gtacgagtcc gtcagcaagt cacaggagtt cttcagcaag 4560
ttcgataaga tttgctacaa tctggacaag ggctacttcg agttctcatt cgattacaag 4620
aacttcggcg acaaggccgc gaagggcaag tggaccattg cgtctttcgg ctcccggctc 4680
atcaacttca ggaatagcga taagaaccac aattgggaca ccagggaggt gtacccgaca 4740
aaggagctgg agaagctgct caaggactac tcaatcgagt acggccatgg cgagtgcatt 4800
aaggccgcga tctgcggcga gagcgataag aagttcttcg ccaagctgac ctcagtcctc 4860
aatacaatcc tgcagatgag gaactctaag accggcacag agctggacta cctgatttcc 4920
ccagtggccg atgtcaacgg caatttcttc gacagccgcc aggcgccgaa gaatatgcca 4980
caggacgccg acgccaacgg cgcataccac atcggcctca agggcctgat gctgctcgga 5040
cgcattaaga acaatcagga gggcaagaag ctcaacctcg tgatcaagaa cgaggagtac 5100
ttcgagttcg tccaaaaccg gaacaattcc ggcggcagcc caaagaagaa gaggaaggtg 5160
agcggcggca gcccaaagaa gaagcgcaag gtctag 5196
<210> 12
<211> 4980
<212> DNA
<213> Artificial Sequence
<220>
<223> dLBCPF1-ABE7.10-2NLS
<400> 12
atgccaaaaa agaagagaaa ggtttcaggc ggctcctccg aggtggagtt ctctcacgag 60
tattggatga ggcacgctct tacacttgct aagagagctt gggacgaaag agaagtgcca 120
gttggcgccg ttcttgtgca taataatagg gtgatcggcg agggttggaa tagaccaatt 180
ggaaggcatg atccaacagc tcacgcagag attatggctc tcagacaagg cggcctcgtt 240
atgcagaact acaggctcat tgacgctaca ctctacgtga cactcgaacc ttgcgttatg 300
tgcgccggag ctatgattca ttctaggatt ggcagggtcg tgtttggagc tagggacgct 360
aaaacaggag ccgccggatc tcttatggac gtgttgcatc atccaggcat gaaccatagg 420
gtggagatta cagagggcat tcttgcagac gagtgcgctg ctcttctttc cgatttcttc 480
aggatgagaa ggcaggagat taaggcccag aagaaggctc agtcttctac agatagcgga 540
ggatcttccg gaggatctag cggctccgag acaccaggaa catccgaaag cgctacacca 600
gaatctagcg gaggctcttc cggaggatct tctgaagtgg agttctccca cgagtattgg 660
atgaggcacg ctcttacact tgctaaaagg gctagggacg aaagggaagt tccagttgga 720
gctgttctcg tgctcaataa cagggtgatt ggcgagggtt ggaatagagc cattggactc 780
catgatccaa cagctcacgc agagattatg gctcttagac aaggcggcct cgttatgcag 840
aattacagac tcatcgacgc cacactctac gttaccttcg aaccttgcgt tatgtgcgcc 900
ggagctatga tccattctag gattggcagg gtcgtgttcg gcgttagaaa cgctaagaca 960
ggagctgcag gctctcttat ggacgttctt cattacccag gcatgaatca tagagtggag 1020
atcacagaag gcattcttgc agacgagtgc gcagctctcc tttgctattt cttcaggatg 1080
ccgaggcaag ttttcaacgc tcagaagaag gcccagtctt ctacagattc cggcggatct 1140
tctggaggat ctagcggctc cgagacacca ggaacatccg aatccgctac accagagtct 1200
tctggaggat ctagcggagg atctcttaag tcaaagctcg agaaattcac caactgttat 1260
tcgttgagca aaacactgcg gtttaaagcg attccagtcg gcaagactca agagaatata 1320
gacaataagc ggctgttggt ggaagatgaa aagcgcgcgg aagactacaa aggggtgaag 1380
aagttgttgg acagatacta cctctctttt atcaatgatg tcttgcactc aatcaaattg 1440
aagaatctga acaactacat ctccctcttc agaaagaaaa caaggacaga aaaggagaat 1500
aaggaacttg aaaatttgga gatcaatctg aggaaagaga tcgcgaaagc ctttaaaggc 1560
aacgaaggat acaaaagtct gttcaagaag gatataattg agacaatttt gccagagttc 1620
ctcgatgaca aggacgagat tgcgctggtc aattcgttca acggattcac aacagcattc 1680
acaggcttct ttgataatcg ggaaaatatg ttctctgagg aggcaaagtc cacttctatt 1740
gcgttcaggt gtatcaatga gaatctcact aggtacattt ccaacatgga tatctttgag 1800
aaggttgacg caatttttga caagcacgaa gttcaggaga ttaaggagaa gatcctcaat 1860
tccgattatg acgttgagga cttcttcgaa ggtgagtttt ttaatttcgt gctcactcaa 1920
gagggtatcg acgtgtataa tgcgatcatc ggtgggttcg tgactgagtc cggtgaaaag 1980
attaagggat tgaacgagta tatcaacctt tacaaccaaa agacgaaaca gaagctgcca 2040
aagttcaagc ctctttacaa acaggttctt tcagaccgcg agtcactctc gttctatggg 2100
gagggctaca cttcggatga ggaagtcctg gaggtgttca ggaatactct caataagaat 2160
tcggagattt tctcttctat aaaaaaactg gaaaagttgt ttaagaattt tgacgaatac 2220
tctagcgccg gcatatttgt gaaaaacggc ccggccatat caacgataag taaagatatc 2280
ttcggcgaat ggaacgtgat cagagacaaa tggaacgcgg agtatgacga tattcacctg 2340
aagaagaagg ctgtcgtaac ggagaagtac gaggatgatc gcaggaaaag cttcaaaaag 2400
atcggaagtt tcagcctgga acagttgcag gagtatgctg acgccgatct tagcgtcgtc 2460
gagaagttga aggagataat catccaaaag gtcgacgaga tatataaagt ctatggatca 2520
agtgaaaaac tgttcgacgc cgacttcgtt ttggagaagt ccctgaagaa gaacgacgct 2580
gttgttgcca ttatgaagga tctgctcgac agcgtgaaga gtttcgagaa ctatattaag 2640
gcttttttcg gggaggggaa ggagactaac agagatgagt ccttctacgg agacttcgtc 2700
ctcgcgtacg atatactcct taaggtagac cacatctacg acgcaatcag aaattacgtg 2760
acacaaaagc cgtacagcaa ggacaagttc aaactctact tccagaaccc ccagttcatg 2820
ggcggctggg acaaggacaa ggaaacggat tacagggcta cgatcctgag gtatggttca 2880
aaatactact tggcgattat ggacaagaag tacgccaagt gtctccagaa gattgacaaa 2940
gacgatgtca atggcaatta tgagaagatc aactacaagc tgcttccggg tccgaacaag 3000
atgctcccaa aggttttctt cagcaagaaa tggatggcct actataaccc aagcgaggac 3060
atccagaaga tttataagaa cggtacgttc aagaagggcg acatgttcaa tcttaacgac 3120
tgtcacaagc tgatcgactt cttcaaagac tcaattagcc ggtacccaaa gtggtctaac 3180
gcctatgact tcaacttttc ggaaaccgag aagtacaagg atatagccgg attttataga 3240
gaggtggaag agcagggcta caaggtgtca ttcgagtccg ccagcaagaa ggaagtggac 3300
aagctcgtgg aagagggtaa gctctacatg ttccagattt ataataaaga ctttagcgat 3360
aagagccacg ggacacctaa tctccacaca atgtatttca agctgctctt cgacgagaat 3420
aaccacggcc aaatcaggtt gtcaggaggg gctgaactct tcatgcggcg cgctagcctt 3480
aagaaggagg agcttgtagt ccaccctgcg aatagtccaa ttgcgaataa gaacccggac 3540
aatcctaaaa agactacaac attgagctac gacgtgtaca aggataagag gttttccgag 3600
gatcagtacg agctccacat cccgattgcg atcaacaagt gcccaaagaa tattttcaag 3660
ataaacacag aggtgcgtgt actcctgaag catgacgaca atccttacgt cattgggatt 3720
gctcggggcg agaggaacct cctctatatt gtggtggtgg acgggaaggg gaacatagtc 3780
gaacagtact cccttaacga aataattaac aatttcaacg gcatccgtat caagaccgac 3840
taccattcgt tgctggacaa gaaggagaag gagagatttg aggcgcggca aaattggaca 3900
agtatcgaga acatcaagga actcaaagca ggttatatct ctcaagttgt gcataagata 3960
tgcgagctgg ttgagaagta tgacgcagtg atcgctcttg aggacctcaa ctcgggcttt 4020
aagaattcta gagttaaagt ggagaagcag gtctatcaaa agttcgagaa gatgcttata 4080
gataagctca actacatggt cgataagaaa tcgaacccat gtgccaccgg cggcgcactc 4140
aaaggttacc aaataacaaa caaattcgag tccttcaaat cgatgagtac tcagaatggg 4200
ttcatatttt atataccggc gtggcttacg tctaagatcg acccgtcaac tggttttgtc 4260
aacctgttga agacgaaata cacgtccatt gccgattcga aaaagttcat atctagtttt 4320
gatcgtatta tgtacgtccc agaggaagat cttttcgagt ttgctctcga ctacaaaaac 4380
ttttcgcgga ccgatgcgga ttacattaaa aaatggaaac tctattcgta cggcaacaga 4440
atcaggattt ttcgcaaccc taagaagaat aacgtctttg attgggagga agtttgcttg 4500
actagcgcgt acaaggagct ctttaataag tatggcatta actaccaaca gggtgatatc 4560
agagcactgc tttgcgaaca atctgacaag gctttctact catccttcat ggctttgatg 4620
agcctgatgc tccagatgag aaattcaatt acaggcagaa ccgacgtgga tttcttgatc 4680
tccccggtta aaaattctga tggcatcttt tacgatagca ggaactatga agcgcaagag 4740
aatgcgattc tgccaaaaaa tgcagacgcc aacggtgcct ataacatcgc caggaaagtc 4800
ctgtgggcga tcggccagtt caaaaaggcc gaagacgaaa aattggacaa ggtcaaaatc 4860
gctatcagca acaaagagtg gctggagtat gctcagacat ccgtaaagca ttccggcggc 4920
agcccaaaga agaagaggaa ggtgagcggc ggcagcccaa agaagaagcg caaggtctag 4980
<210> 13
<211> 6506
<212> DNA
<213> Artificial Sequence
<220>
<223> dLBCPF1-ABE2-X
<400> 13
ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60
agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120
tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180
tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240
gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300
ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360
gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420
agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480
taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540
aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600
tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 660
cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 720
ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 780
gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 840
caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900
ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960
tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020
taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080
atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140
cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200
ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 1260
gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1320
cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1380
acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1440
ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1500
tctgcaggtc gaagcttgaa gcaaacatgc caaaaaagaa gagaaaggtt tcaggcggct 1560
cctccgaggt ggagttctct cacgagtatt ggatgaggca cgctcttaca cttgctaaga 1620
gagcttggga cgaaagagaa gtgccagttg gcgccgttct tgtgcataat aatagggtga 1680
tcggcgaggg ttggaataga ccaattggaa ggcatgatcc aacagctcac gcagagatta 1740
tggctctcag acaaggcggc ctcgttatgc agaactacag gctcattgac gctacactct 1800
acgtgacact cgaaccttgc gttatgtgcg ccggagctat gattcattct aggattggca 1860
gggtcgtgtt tggagctagg gacgctaaaa caggagccgc cggatctctt atggacgtgt 1920
tgcatcatcc aggcatgaac catagggtgg agattacaga gggcattctt gcagacgagt 1980
gcgctgctct tctttccgat ttcttcagga tgagaaggca ggagattaag gcccagaaga 2040
aggctcagtc ttctacagat agcggaggat cttccggagg atctagcggc tccgagacac 2100
caggaacatc cgaaagcgct acaccagaat ctagcggagg ctcttccgga ggatcttctg 2160
aagtggagtt ctcccacgag tattggatga ggcacgctct tacacttgct aaaagggcta 2220
gggacgaaag ggaagttcca gttggagctg ttctcgtgct caataacagg gtgattggcg 2280
agggttggaa tagagccatt ggactccatg atccaacagc tcacgcagag attatggctc 2340
ttagacaagg cggcctcgtt atgcagaatt acagactcat cgacgccaca ctctacgtta 2400
ccttcgaacc ttgcgttatg tgcgccggag ctatgatcca ttctaggatt ggcagggtcg 2460
tgttcggcgt tagaaacgct aagacaggag ctgcaggctc tcttatggac gttcttcatt 2520
acccaggcat gaatcataga gtggagatca cagaaggcat tcttgcagac gagtgcgcag 2580
ctctcctttg ctatttcttc aggatgccga ggcaagtttt caacgctcag aagaaggccc 2640
agtcttctac agattccggc ggatcttctg gaggatctag cggctccgag acaccaggaa 2700
catccgaatc cgctacacca gagtcttctg gaggatctag cggaggatct cttaagtcaa 2760
agctcgagaa attcaccaac tgttattcgt tgagcaaaac actgcggttt aaagcgattc 2820
cagtcggcaa gactcaagag aatatagaca ataagcggct gttggtggaa gatgaaaagc 2880
gcgcggaaga ctacaaaggg gtgaagaagt tgttggacag atactacctc tcttttatca 2940
atgatgtctt gcactcaatc aaattgaaga atctgaacaa ctacatctcc ctcttcagaa 3000
agaaaacaag gacagaaaag gagaataagg aacttgaaaa tttggagatc aatctgagga 3060
aagagatcgc gaaagccttt aaaggcaacg aaggatacaa aagtctgttc aagaaggata 3120
taattgagac aattttgcca gagttcctcg atgacaagga cgagattgcg ctggtcaatt 3180
cgttcaacgg attcacaaca gcattcacag gcttctttga taatcgggaa aatatgttct 3240
ctgaggaggc aaagtccact tctattgcgt tcaggtgtat caatgagaat ctcactaggt 3300
acatttccaa catggatatc tttgagaagg ttgacgcaat ttttgacaag cacgaagttc 3360
aggagattaa ggagaagatc ctcaattccg attatgacgt tgaggacttc ttcgaaggtg 3420
agttttttaa tttcgtgctc actcaagagg gtatcgacgt gtataatgcg atcatcggtg 3480
ggttcgtgac tgagtccggt gaaaagatta agggattgaa cgagtatatc aacctttaca 3540
accaaaagac gaaacagaag ctgccaaagt tcaagcctct ttacaaacag gttctttcag 3600
accgcgagtc actctcgttc tatggggagg gctacacttc ggatgaggaa gtcctggagg 3660
tgttcaggaa tactctcaat aagaattcgg agattttctc ttctataaaa aaactggaaa 3720
agttgtttaa gaattttgac gaatactcta gcgccggcat atttgtgaaa aacggcccgg 3780
ccatatcaac gataagtaaa gatatcttcg gcgaatggaa cgtgatcaga gacaaatgga 3840
acgcggagta tgacgatatt cacctgaaga agaaggctgt cgtaacggag aagtacgagg 3900
atgatcgcag gaaaagcttc aaaaagatcg gaagtttcag cctggaacag ttgcaggagt 3960
atgctgacgc cgatcttagc gtcgtcgaga agttgaagga gataatcatc caaaaggtcg 4020
acgagatata taaagtctat ggatcaagtg aaaaactgtt cgacgccgac ttcgttttgg 4080
agaagtccct gaagaagaac gacgctgttg ttgccattat gaaggatctg ctcgacagcg 4140
tgaagagttt cgagaactat attaaggctt ttttcgggga ggggaaggag actaacagag 4200
atgagtcctt ctacggagac ttcgtcctcg cgtacgatat actccttaag gtagaccaca 4260
tctacgacgc aatcagaaat tacgtgacac aaaagccgta cagcaaggac aagttcaaac 4320
tctacttcca gaacccccag ttcatgggcg gctgggacaa ggacaaggaa acggattaca 4380
gggctacgat cctgaggtat ggttcaaaat actacttggc gattatggac aagaagtacg 4440
ccaagtgtct ccagaagatt gacaaagacg atgtcaatgg caattatgag aagatcaact 4500
acaagctgct tccgggtccg aacaagatgc tcccaaaggt tttcttcagc aagaaatgga 4560
tggcctacta taacccaagc gaggacatcc agaagattta taagaacggt acgttcaaga 4620
agggcgacat gttcaatctt aacgactgtc acaagctgat cgacttcttc aaagactcaa 4680
ttagccggta cccaaagtgg tctaacgcct atgacttcaa cttttcggaa accgagaagt 4740
acaaggatat agccggattt tatagagagg tggaagagca gggctacaag gtgtcattcg 4800
agtccgccag caagaaggaa gtggacaagc tcgtggaaga gggtaagctc tacatgttcc 4860
agatttataa taaagacttt agcgataaga gccacgggac acctaatctc cacacaatgt 4920
atttcaagct gctcttcgac gagaataacc acggccaaat caggttgtca ggaggggctg 4980
aactcttcat gcggcgcgct agccttaaga aggaggagct tgtagtccac cctgcgaata 5040
gtccaattgc gaataagaac ccggacaatc ctaaaaagac tacaacattg agctacgacg 5100
tgtacaagga taagaggttt tccgaggatc agtacgagct ccacatcccg attgcgatca 5160
acaagtgccc aaagaatatt ttcaagataa acacagaggt gcgtgtactc ctgaagcatg 5220
acgacaatcc ttacgtcatt gggattgctc ggggcgagag gaacctcctc tatattgtgg 5280
tggtggacgg gaaggggaac atagtcgaac agtactccct taacgaaata attaacaatt 5340
tcaacggcat ccgtatcaag accgactacc attcgttgct ggacaagaag gagaaggaga 5400
gatttgaggc gcggcaaaat tggacaagta tcgagaacat caaggaactc aaagcaggtt 5460
atatctctca agttgtgcat aagatatgcg agctggttga gaagtatgac gcagtgatcg 5520
ctcttgagga cctcaactcg ggctttaaga attctagagt taaagtggag aagcaggtct 5580
atcaaaagtt cgagaagatg cttatagata agctcaacta catggtcgat aagaaatcga 5640
acccatgtgc caccggcggc gcactcaaag gttaccaaat aacaaacaaa ttcgagtcct 5700
tcaaatcgat gagtactcag aatgggttca tattttatat accggcgtgg cttacgtcta 5760
agatcgaccc gtcaactggt tttgtcaacc tgttgaagac gaaatacacg tccattgccg 5820
attcgaaaaa gttcatatct agttttgatc gtattatgta cgtcccagag gaagatcttt 5880
tcgagtttgc tctcgactac aaaaactttt cgcggaccga tgcggattac attaaaaaat 5940
ggaaactcta ttcgtacggc aacagaatca ggatttttcg caaccctaag aagaataacg 6000
tctttgattg ggaggaagtt tgcttgacta gcgcgtacaa ggagctcttt aataagtatg 6060
gcattaacta ccaacagggt gatatcagag cactgctttg cgaacaatct gacaaggctt 6120
tctactcatc cttcatggct ttgatgagcc tgatgctcca gatgagaaat tcaattacag 6180
gcagaaccga cgtggatttc ttgatctccc cggttaaaaa ttctgatggc atcttttacg 6240
atagcaggaa ctatgaagcg caagagaatg cgattctgcc aaaaaatgca gacgccaacg 6300
gtgcctataa catcgccagg aaagtcctgt gggcgatcgg ccagttcaaa aaggccgaag 6360
acgaaaaatt ggacaaggtc aaaatcgcta tcagcaacaa agagtggctg gagtatgctc 6420
agacatccgt aaagcattcc ggcggcagcc caaagaagaa gaggaaggtg agcggcggca 6480
gcccaaagaa gaagcgcaag gtctag 6506
<210> 14
<211> 4971
<212> DNA
<213> Artificial Sequence
<220>
<223> LBCPF1-ABE2-X2
<400> 14
atgtcaaagc tcgagaaatt caccaactgt tattcgttga gcaaaacact gcggtttaaa 60
gcgattccag tcggcaagac tcaagagaat atagacaata agcggctgtt ggtggaagat 120
gaaaagcgcg cggaagacta caaaggggtg aagaagttgt tggacagata ctacctctct 180
tttatcaatg atgtcttgca ctcaatcaaa ttgaagaatc tgaacaacta catctccctc 240
ttcagaaaga aaacaaggac agaaaaggag aataaggaac ttgaaaattt ggagatcaat 300
ctgaggaaag agatcgcgaa agcctttaaa ggcaacgaag gatacaaaag tctgttcaag 360
aaggatataa ttgagacaat tttgccagag ttcctcgatg acaaggacga gattgcgctg 420
gtcaattcgt tcaacggatt cacaacagca ttcacaggct tctttgataa tcgggaaaat 480
atgttctctg aggaggcaaa gtccacttct attgcgttca ggtgtatcaa tgagaatctc 540
actaggtaca tttccaacat ggatatcttt gagaaggttg acgcaatttt tgacaagcac 600
gaagttcagg agattaagga gaagatcctc aattccgatt atgacgttga ggacttcttc 660
gaaggtgagt tttttaattt cgtgctcact caagagggta tcgacgtgta taatgcgatc 720
atcggtgggt tcgtgactga gtccggtgaa aagattaagg gattgaacga gtatatcaac 780
ctttacaacc aaaagacgaa acagaagctg ccaaagttca agcctcttta caaacaggtt 840
ctttcagacc gcgagtcact ctcgttctat ggggagggct acacttcgga tgaggaagtc 900
ctggaggtgt tcaggaatac tctcaataag aattcggaga ttttctcttc tataaaaaaa 960
ctggaaaagt tgtttaagaa ttttgacgaa tactctagcg ccggcatatt tgtgaaaaac 1020
ggcccggcca tatcaacgat aagtaaagat atcttcggcg aatggaacgt gatcagagac 1080
aaatggaacg cggagtatga cgatattcac ctgaagaaga aggctgtcgt aacggagaag 1140
tacgaggatg atcgcaggaa aagcttcaaa aagatcggaa gtttcagcct ggaacagttg 1200
caggagtatg ctgacgccga tcttagcgtc gtcgagaagt tgaaggagat aatcatccaa 1260
aaggtcgacg agatatataa agtctatgga tcaagtgaaa aactgttcga cgccgacttc 1320
gttttggaga agtccctgaa gaagaacgac gctgttgttg ccattatgaa ggatctgctc 1380
gacagcgtga agagtttcga gaactatatt aaggcttttt tcggggaggg gaaggagact 1440
aacagagatg agtccttcta cggagacttc gtcctcgcgt acgatatact ccttaaggta 1500
gaccacatct acgacgcaat cagaaattac gtgacacaaa agccgtacag caaggacaag 1560
ttcaaactct acttccagaa cccccagttc atgggcggct gggacaagga caaggaaacg 1620
gattacaggg ctacgatcct gaggtatggt tcaaaatact acttggcgat tatggacaag 1680
aagtacgcca agtgtctcca gaagattgac aaagacgatg tcaatggcaa ttatgagaag 1740
atcaactaca agctgcttcc gggtccgaac aagatgctcc caaaggtttt cttcagcaag 1800
aaatggatgg cctactataa cccaagcgag gacatccaga agatttataa gaacggtacg 1860
ttcaagaagg gcgacatgtt caatcttaac gactgtcaca agctgatcga cttcttcaaa 1920
gactcaatta gccggtaccc aaagtggtct aacgcctatg acttcaactt ttcggaaacc 1980
gagaagtaca aggatatagc cggattttat agagaggtgg aagagcaggg ctacaaggtg 2040
tcattcgagt ccgccagcaa gaaggaagtg gacaagctcg tggaagaggg taagctctac 2100
atgttccaga tttataataa agactttagc gataagagcc acgggacacc taatctccac 2160
acaatgtatt tcaagctgct cttcgacgag aataaccacg gccaaatcag gttgtcagga 2220
ggggctgaac tcttcatgcg gcgcgctagc cttaagaagg aggagcttgt agtccaccct 2280
gcgaatagtc caattgcgaa taagaacccg gacaatccta aaaagactac aacattgagc 2340
tacgacgtgt acaaggataa gaggttttcc gaggatcagt acgagctcca catcccgatt 2400
gcgatcaaca agtgcccaaa gaatattttc aagataaaca cagaggtgcg tgtactcctg 2460
aagcatgacg acaatcctta cgtcattggg attgctcggg gcgagaggaa cctcctctat 2520
attgtggtgg tggacgggaa ggggaacata gtcgaacagt actcccttaa cgaaataatt 2580
aacaatttca acggcatccg tatcaagacc gactaccatt cgttgctgga caagaaggag 2640
aaggagagat ttgaggcgcg gcaaaattgg acaagtatcg agaacatcaa ggaactcaaa 2700
gcaggttata tctctcaagt tgtgcataag atatgcgagc tggttgagaa gtatgacgca 2760
gtgatcgctc ttgaggacct caactcgggc tttaagaatt ctagagttaa agtggagaag 2820
caggtctatc aaaagttcga gaagatgctt atagataagc tcaactacat ggtcgataag 2880
aaatcgaacc catgtgccac cggcggcgca ctcaaaggtt accaaataac aaacaaattc 2940
gagtccttca aatcgatgag tactcagaat gggttcatat tttatatacc ggcgtggctt 3000
acgtctaaga tcgacccgtc aactggtttt gtcaacctgt tgaagacgaa atacacgtcc 3060
attgccgatt cgaaaaagtt catatctagt tttgatcgta ttatgtacgt cccagaggaa 3120
gatcttttcg agtttgctct cgactacaaa aacttttcgc ggaccgatgc ggattacatt 3180
aaaaaatgga aactctattc gtacggcaac agaatcagga tttttcgcaa ccctaagaag 3240
aataacgtct ttgattggga ggaagtttgc ttgactagcg cgtacaagga gctctttaat 3300
aagtatggca ttaactacca acagggtgat atcagagcac tgctttgcga acaatctgac 3360
aaggctttct actcatcctt catggctttg atgagcctga tgctccagat gagaaattca 3420
attacaggca gaaccgacgt ggatttcttg atctccccgg ttaaaaattc tgatggcatc 3480
ttttacgata gcaggaacta tgaagcgcaa gagaatgcga ttctgccaaa aaatgcagac 3540
gccaacggtg cctataacat cgccaggaaa gtcctgtggg cgatcggcca gttcaaaaag 3600
gccgaagacg aaaaattgga caaggtcaaa atcgctatca gcaacaaaga gtggctggag 3660
tatgctcaga catccgtaaa gcataagctt atgccaaaaa agaagagaaa ggtttcaggc 3720
ggctcctccg aggtggagtt ctctcacgag tattggatga ggcacgctct tacacttgct 3780
aagagagctt gggacgaaag agaagtgcca gttggcgccg ttcttgtgca taataatagg 3840
gtgatcggcg agggttggaa tagaccaatt ggaaggcatg atccaacagc tcacgcagag 3900
attatggctc tcagacaagg cggcctcgtt atgcagaact acaggctcat tgacgctaca 3960
ctctacgtga cactcgaacc ttgcgttatg tgcgccggag ctatgattca ttctaggatt 4020
ggcagggtcg tgtttggagc tagggacgct aaaacaggag ccgccggatc tcttatggac 4080
gtgttgcatc atccaggcat gaaccatagg gtggagatta cagagggcat tcttgcagac 4140
gagtgcgctg ctcttctttc cgatttcttc aggatgagaa ggcaggagat taaggcccag 4200
aagaaggctc agtcttctac agatagcgga ggatcttccg gaggatctag cggctccgag 4260
acaccaggaa catccgaaag cgctacacca gaatctagcg gaggctcttc cggaggatct 4320
tctgaagtgg agttctccca cgagtattgg atgaggcacg ctcttacact tgctaaaagg 4380
gctagggacg aaagggaagt tccagttgga gctgttctcg tgctcaataa cagggtgatt 4440
ggcgagggtt ggaatagagc cattggactc catgatccaa cagctcacgc agagattatg 4500
gctcttagac aaggcggcct cgttatgcag aattacagac tcatcgacgc cacactctac 4560
gttaccttcg aaccttgcgt tatgtgcgcc ggagctatga tccattctag gattggcagg 4620
gtcgtgttcg gcgttagaaa cgctaagaca ggagctgcag gctctcttat ggacgttctt 4680
cattacccag gcatgaatca tagagtggag atcacagaag gcattcttgc agacgagtgc 4740
gcagctctcc tttgctattt cttcaggatg ccgaggcaag ttttcaacgc tcagaagaag 4800
gcccagtctt ctacagattc cggcggatct tctggaggat ctagcggctc cgagacacca 4860
ggaacatccg aatccgctac accagagtct tctggaggat ctagcggagg atctcttaag 4920
aagagaccag cagctacaaa gaaggccgga caagctaaga agaagaagta g 4971
<210> 15
<211> 4992
<212> DNA
<213> Artificial Sequence
<220>
<223> LBCPF1-ABE2-X3
<400> 15
atgccaaaaa agaagagaaa ggtttcaaag ctcgagaaat tcaccaactg ttattcgttg 60
agcaaaacac tgcggtttaa agcgattcca gtcggcaaga ctcaagagaa tatagacaat 120
aagcggctgt tggtggaaga tgaaaagcgc gcggaagact acaaaggggt gaagaagttg 180
ttggacagat actacctctc ttttatcaat gatgtcttgc actcaatcaa attgaagaat 240
ctgaacaact acatctccct cttcagaaag aaaacaagga cagaaaagga gaataaggaa 300
cttgaaaatt tggagatcaa tctgaggaaa gagatcgcga aagcctttaa aggcaacgaa 360
ggatacaaaa gtctgttcaa gaaggatata attgagacaa ttttgccaga gttcctcgat 420
gacaaggacg agattgcgct ggtcaattcg ttcaacggat tcacaacagc attcacaggc 480
ttctttgata atcgggaaaa tatgttctct gaggaggcaa agtccacttc tattgcgttc 540
aggtgtatca atgagaatct cactaggtac atttccaaca tggatatctt tgagaaggtt 600
gacgcaattt ttgacaagca cgaagttcag gagattaagg agaagatcct caattccgat 660
tatgacgttg aggacttctt cgaaggtgag ttttttaatt tcgtgctcac tcaagagggt 720
atcgacgtgt ataatgcgat catcggtggg ttcgtgactg agtccggtga aaagattaag 780
ggattgaacg agtatatcaa cctttacaac caaaagacga aacagaagct gccaaagttc 840
aagcctcttt acaaacaggt tctttcagac cgcgagtcac tctcgttcta tggggagggc 900
tacacttcgg atgaggaagt cctggaggtg ttcaggaata ctctcaataa gaattcggag 960
attttctctt ctataaaaaa actggaaaag ttgtttaaga attttgacga atactctagc 1020
gccggcatat ttgtgaaaaa cggcccggcc atatcaacga taagtaaaga tatcttcggc 1080
gaatggaacg tgatcagaga caaatggaac gcggagtatg acgatattca cctgaagaag 1140
aaggctgtcg taacggagaa gtacgaggat gatcgcagga aaagcttcaa aaagatcgga 1200
agtttcagcc tggaacagtt gcaggagtat gctgacgccg atcttagcgt cgtcgagaag 1260
ttgaaggaga taatcatcca aaaggtcgac gagatatata aagtctatgg atcaagtgaa 1320
aaactgttcg acgccgactt cgttttggag aagtccctga agaagaacga cgctgttgtt 1380
gccattatga aggatctgct cgacagcgtg aagagtttcg agaactatat taaggctttt 1440
ttcggggagg ggaaggagac taacagagat gagtccttct acggagactt cgtcctcgcg 1500
tacgatatac tccttaaggt agaccacatc tacgacgcaa tcagaaatta cgtgacacaa 1560
aagccgtaca gcaaggacaa gttcaaactc tacttccaga acccccagtt catgggcggc 1620
tgggacaagg acaaggaaac ggattacagg gctacgatcc tgaggtatgg ttcaaaatac 1680
tacttggcga ttatggacaa gaagtacgcc aagtgtctcc agaagattga caaagacgat 1740
gtcaatggca attatgagaa gatcaactac aagctgcttc cgggtccgaa caagatgctc 1800
ccaaaggttt tcttcagcaa gaaatggatg gcctactata acccaagcga ggacatccag 1860
aagatttata agaacggtac gttcaagaag ggcgacatgt tcaatcttaa cgactgtcac 1920
aagctgatcg acttcttcaa agactcaatt agccggtacc caaagtggtc taacgcctat 1980
gacttcaact tttcggaaac cgagaagtac aaggatatag ccggatttta tagagaggtg 2040
gaagagcagg gctacaaggt gtcattcgag tccgccagca agaaggaagt ggacaagctc 2100
gtggaagagg gtaagctcta catgttccag atttataata aagactttag cgataagagc 2160
cacgggacac ctaatctcca cacaatgtat ttcaagctgc tcttcgacga gaataaccac 2220
ggccaaatca ggttgtcagg aggggctgaa ctcttcatgc ggcgcgctag ccttaagaag 2280
gaggagcttg tagtccaccc tgcgaatagt ccaattgcga ataagaaccc ggacaatcct 2340
aaaaagacta caacattgag ctacgacgtg tacaaggata agaggttttc cgaggatcag 2400
tacgagctcc acatcccgat tgcgatcaac aagtgcccaa agaatatttt caagataaac 2460
acagaggtgc gtgtactcct gaagcatgac gacaatcctt acgtcattgg gattgctcgg 2520
ggcgagagga acctcctcta tattgtggtg gtggacggga aggggaacat agtcgaacag 2580
tactccctta acgaaataat taacaatttc aacggcatcc gtatcaagac cgactaccat 2640
tcgttgctgg acaagaagga gaaggagaga tttgaggcgc ggcaaaattg gacaagtatc 2700
gagaacatca aggaactcaa agcaggttat atctctcaag ttgtgcataa gatatgcgag 2760
ctggttgaga agtatgacgc agtgatcgct cttgaggacc tcaactcggg ctttaagaat 2820
tctagagtta aagtggagaa gcaggtctat caaaagttcg agaagatgct tatagataag 2880
ctcaactaca tggtcgataa gaaatcgaac ccatgtgcca ccggcggcgc actcaaaggt 2940
taccaaataa caaacaaatt cgagtccttc aaatcgatga gtactcagaa tgggttcata 3000
ttttatatac cggcgtggct tacgtctaag atcgacccgt caactggttt tgtcaacctg 3060
ttgaagacga aatacacgtc cattgccgat tcgaaaaagt tcatatctag ttttgatcgt 3120
attatgtacg tcccagagga agatcttttc gagtttgctc tcgactacaa aaacttttcg 3180
cggaccgatg cggattacat taaaaaatgg aaactctatt cgtacggcaa cagaatcagg 3240
atttttcgca accctaagaa gaataacgtc tttgattggg aggaagtttg cttgactagc 3300
gcgtacaagg agctctttaa taagtatggc attaactacc aacagggtga tatcagagca 3360
ctgctttgcg aacaatctga caaggctttc tactcatcct tcatggcttt gatgagcctg 3420
atgctccaga tgagaaattc aattacaggc agaaccgacg tggatttctt gatctccccg 3480
gttaaaaatt ctgatggcat cttttacgat agcaggaact atgaagcgca agagaatgcg 3540
attctgccaa aaaatgcaga cgccaacggt gcctataaca tcgccaggaa agtcctgtgg 3600
gcgatcggcc agttcaaaaa ggccgaagac gaaaaattgg acaaggtcaa aatcgctatc 3660
agcaacaaag agtggctgga gtatgctcag acatccgtaa agcataagct tatgccaaaa 3720
aagaagagaa aggtttcagg cggctcctcc gaggtggagt tctctcacga gtattggatg 3780
aggcacgctc ttacacttgc taagagagct tgggacgaaa gagaagtgcc agttggcgcc 3840
gttcttgtgc ataataatag ggtgatcggc gagggttgga atagaccaat tggaaggcat 3900
gatccaacag ctcacgcaga gattatggct ctcagacaag gcggcctcgt tatgcagaac 3960
tacaggctca ttgacgctac actctacgtg acactcgaac cttgcgttat gtgcgccgga 4020
gctatgattc attctaggat tggcagggtc gtgtttggag ctagggacgc taaaacagga 4080
gccgccggat ctcttatgga cgtgttgcat catccaggca tgaaccatag ggtggagatt 4140
acagagggca ttcttgcaga cgagtgcgct gctcttcttt ccgatttctt caggatgaga 4200
aggcaggaga ttaaggccca gaagaaggct cagtcttcta cagatagcgg aggatcttcc 4260
ggaggatcta gcggctccga gacaccagga acatccgaaa gcgctacacc agaatctagc 4320
ggaggctctt ccggaggatc ttctgaagtg gagttctccc acgagtattg gatgaggcac 4380
gctcttacac ttgctaaaag ggctagggac gaaagggaag ttccagttgg agctgttctc 4440
gtgctcaata acagggtgat tggcgagggt tggaatagag ccattggact ccatgatcca 4500
acagctcacg cagagattat ggctcttaga caaggcggcc tcgttatgca gaattacaga 4560
ctcatcgacg ccacactcta cgttaccttc gaaccttgcg ttatgtgcgc cggagctatg 4620
atccattcta ggattggcag ggtcgtgttc ggcgttagaa acgctaagac aggagctgca 4680
ggctctctta tggacgttct tcattaccca ggcatgaatc atagagtgga gatcacagaa 4740
ggcattcttg cagacgagtg cgcagctctc ctttgctatt tcttcaggat gccgaggcaa 4800
gttttcaacg ctcagaagaa ggcccagtct tctacagatt ccggcggatc ttctggagga 4860
tctagcggct ccgagacacc aggaacatcc gaatccgcta caccagagtc ttctggagga 4920
tctagcggag gatctcttaa gaagagacca gcagctacaa agaaggccgg acaagctaag 4980
aagaagaagt ag 4992
<210> 16
<211> 5686
<212> DNA
<213> Artificial Sequence
<220>
<223> PJIT163-GFP
<400> 16
gagctcggta cctgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60
gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120
ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180
atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240
tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300
tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360
tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420
gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat 480
aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540
actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600
gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660
ggcacggcat ctctgtcgct gcctctggac ccctctcgat cgagagttcc gctccaccgt 720
tggacttgct ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg 780
cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc 840
accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc 900
tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat 960
ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccccctctct 1020
accttctcta gatcggcgtt ccggtccatg gttagggccc ggtagttcta cttctgttca 1080
tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc 1140
gacctgtacg tcagacacgt tctgattgct aacttgccag tgtttctctt tggggaatcc 1200
tgggatggct ctagccgttc cgcagacggg atcgatttca tgattttttt tgtttcgttg 1260
catagggttt ggtttgccct tttcctttat ttcaatatat gccgtgcact tgtttgtcgg 1320
gtcatctttt catgcttttt tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg 1380
ttctagatcg gagtagaatt aattctgttt caaactacct ggtggattta ttaattttgg 1440
atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg gatggaaata 1500
tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata cagagatgct 1560
ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt cgttctagat 1620
cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa ctgtatgtgt 1680
gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat ctaggatagg 1740
tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc agcatctatt 1800
catatgctct aaccttgagt acctatctat tataataaac aagtatgttt tataattatt 1860
ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat ttttttagcc 1920
ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt 1980
gtttggtgtt acttctgcaa agcttgtcga cggatccatg gtgagcaagg gcgaggagct 2040
gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 2100
cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 2160
ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg 2220
cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 2280
catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 2340
gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 2400
catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 2460
ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 2520
ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 2580
catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 2640
gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 2700
cgggatcact cacggcatgg acgagctgta caagtaaccc gggaattcgg tacgctgaaa 2760
tcaccagtct ctctctacaa atctatctct ctctattttc tccataaata atgtgtgagt 2820
agtttcccga taagggaaat tagggttctt atagggtttc gctcatgtgt tgagcatata 2880
agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc 2940
ctaaaaccaa aatccagtac taaaatccag atctcctaaa gtccctatag atctttgtcg 3000
tgaatataaa ccagacacga gacgactaaa cctggagccc agacgccgtt cgaagctaga 3060
agtaccgctt aggcaggagg ccgttaggga aaagatgcta aggcagggtt ggttacgttg 3120
actcccccgt aggtttggtt taaatatgat gaagtggacg gaaggaagga ggaagacaag 3180
gaaggataag gttgcaggcc ctgtgcaagg taagaagatg gaaatttgat agaggtacgc 3240
tactatactt atactatacg ctaagggaat gcttgtattt ataccctata ccccctaata 3300
accccttatc aatttaagaa ataatccgca taagcccccg cttaaaaatt ggtatcagag 3360
ccatgaatag gtctatgacc aaaactcaag aggataaaac ctcaccaaaa tacgaaagag 3420
ttcttaactc taaagataaa agatctttca agatcaaaac tagttccctc acaccggagc 3480
atgcgatatc ctcgagagat ctaggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 3540
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 3600
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 3660
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 3720
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 3780
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 3840
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3900
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3960
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 4020
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 4080
tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 4140
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4200
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4260
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4320
ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 4380
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4440
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4500
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4560
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 4620
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 4680
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 4740
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 4800
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 4860
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 4920
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 4980
gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 5040
tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 5100
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 5160
gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 5220
actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 5280
tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 5340
attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 5400
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 5460
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 5520
aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 5580
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 5640
cgcacatttc cccgaaaagt gccacctgcc agtgccaagc taattc 5686
<210> 17
<211> 720
<212> DNA
<213> Artificial Sequence
<220>
<223> pBUI-mGFP
<400> 17
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccttcaccta cggcgtgtag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actcacggca tggacgagct gtacaagtaa 720
<210> 18
<211> 1307
<212> PRT
<213> Acidaminococcus sp.
<400> 18
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu
1010 1015 1020
Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly
1025 1030 1035
Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala
1040 1045 1050
Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro
1055 1060 1065
Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe
1070 1075 1080
Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu
1085 1090 1095
Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe
1100 1105 1110
Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly
1115 1120 1125
Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn
1130 1135 1140
Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys
1145 1150 1155
Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr
1160 1165 1170
Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu
1175 1180 1185
Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu
1190 1195 1200
Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu
1205 1210 1215
Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly
1220 1225 1230
Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp
1250 1255 1260
Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu
1265 1270 1275
Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile
1280 1285 1290
Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn
1295 1300 1305
<210> 19
<211> 1300
<212> PRT
<213> Francisella novicida
<400> 19
Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys
20 25 30
Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys
35 40 45
Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu
50 55 60
Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser
65 70 75 80
Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys
85 90 95
Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr
100 105 110
Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile
115 120 125
Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln
130 135 140
Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr
145 150 155 160
Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr
165 170 175
Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser
180 185 190
Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu
195 200 205
Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys
210 215 220
Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu
225 230 235 240
Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg
245 250 255
Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr
260 265 270
Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys
275 280 285
Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile
290 295 300
Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys
305 310 315 320
Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser
325 330 335
Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met
340 345 350
Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys
355 360 365
Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln
370 375 380
Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr
385 390 395 400
Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala
405 410 415
Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn
420 425 430
Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala
435 440 445
Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn
450 455 460
Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala
465 470 475 480
Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys
485 490 495
Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys
500 505 510
Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp
515 520 525
Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His
530 535 540
Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His
545 550 555 560
Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val
565 570 575
Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser
580 585 590
Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly
595 600 605
Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys
610 615 620
Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile
625 630 635 640
Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys
645 650 655
Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val
660 665 670
Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile
675 680 685
Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln
690 695 700
Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe
705 710 715 720
Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp
725 730 735
Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu
740 745 750
Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn
755 760 765
Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr
770 775 780
Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg
785 790 795 800
Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn
805 810 815
Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr
820 825 830
Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala
835 840 845
Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu
850 855 860
Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe
865 870 875 880
His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe
885 890 895
Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His
900 905 910
Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu
915 920 925
Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile
930 935 940
Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile
945 950 955 960
Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn
965 970 975
Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile
980 985 990
Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu
995 1000 1005
Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val
1010 1015 1020
Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu
1025 1030 1035
Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg
1040 1045 1050
Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly
1055 1060 1065
Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser
1070 1075 1080
Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys
1085 1090 1095
Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp
1100 1105 1110
Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe
1115 1120 1125
Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr
1130 1135 1140
Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp
1145 1150 1155
Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu
1160 1165 1170
Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly
1175 1180 1185
Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe
1190 1195 1200
Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg
1205 1210 1215
Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val
1220 1225 1230
Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys
1235 1240 1245
Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly
1250 1255 1260
Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu
1265 1270 1275
Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu
1280 1285 1290
Phe Val Gln Asn Arg Asn Asn
1295 1300
<210> 20
<211> 1228
<212> PRT
<213> Lachnospiraceae bacterium
<400> 20
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile
165 170 175
Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys
180 185 190
Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys
195 200 205
Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe
210 215 220
Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile
225 230 235 240
Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn
245 250 255
Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys
260 265 270
Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser
275 280 285
Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe
290 295 300
Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys
305 310 315 320
Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile
325 330 335
Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe
340 345 350
Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp
355 360 365
Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp
370 375 380
Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu
385 390 395 400
Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu
405 410 415
Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser
420 425 430
Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys
435 440 445
Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys
450 455 460
Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr
465 470 475 480
Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
485 490 495
Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr
500 505 510
Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 21
<211> 1329
<212> PRT
<213> Artificial Sequence
<220>
<223> ASCPF1-2NLS
<400> 21
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu
1010 1015 1020
Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly
1025 1030 1035
Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala
1040 1045 1050
Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro
1055 1060 1065
Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe
1070 1075 1080
Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu
1085 1090 1095
Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe
1100 1105 1110
Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly
1115 1120 1125
Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn
1130 1135 1140
Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys
1145 1150 1155
Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr
1160 1165 1170
Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu
1175 1180 1185
Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu
1190 1195 1200
Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu
1205 1210 1215
Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly
1220 1225 1230
Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp
1250 1255 1260
Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu
1265 1270 1275
Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile
1280 1285 1290
Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Ser
1295 1300 1305
Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro
1310 1315 1320
Lys Lys Lys Arg Lys Val
1325
<210> 22
<211> 1322
<212> PRT
<213> Artificial Sequence
<220>
<223> FNCPF1-2NLS
<400> 22
Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys
20 25 30
Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys
35 40 45
Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu
50 55 60
Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser
65 70 75 80
Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys
85 90 95
Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr
100 105 110
Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile
115 120 125
Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln
130 135 140
Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr
145 150 155 160
Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr
165 170 175
Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser
180 185 190
Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu
195 200 205
Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys
210 215 220
Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu
225 230 235 240
Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg
245 250 255
Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr
260 265 270
Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys
275 280 285
Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile
290 295 300
Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys
305 310 315 320
Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser
325 330 335
Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met
340 345 350
Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys
355 360 365
Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln
370 375 380
Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr
385 390 395 400
Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala
405 410 415
Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn
420 425 430
Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala
435 440 445
Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn
450 455 460
Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala
465 470 475 480
Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys
485 490 495
Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys
500 505 510
Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp
515 520 525
Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His
530 535 540
Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His
545 550 555 560
Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val
565 570 575
Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser
580 585 590
Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly
595 600 605
Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys
610 615 620
Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile
625 630 635 640
Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys
645 650 655
Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val
660 665 670
Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile
675 680 685
Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln
690 695 700
Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe
705 710 715 720
Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp
725 730 735
Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu
740 745 750
Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn
755 760 765
Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr
770 775 780
Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg
785 790 795 800
Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn
805 810 815
Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr
820 825 830
Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala
835 840 845
Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu
850 855 860
Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe
865 870 875 880
His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe
885 890 895
Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His
900 905 910
Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu
915 920 925
Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile
930 935 940
Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile
945 950 955 960
Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn
965 970 975
Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile
980 985 990
Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu
995 1000 1005
Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val
1010 1015 1020
Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu
1025 1030 1035
Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg
1040 1045 1050
Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly
1055 1060 1065
Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser
1070 1075 1080
Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys
1085 1090 1095
Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp
1100 1105 1110
Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe
1115 1120 1125
Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr
1130 1135 1140
Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp
1145 1150 1155
Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu
1160 1165 1170
Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly
1175 1180 1185
Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe
1190 1195 1200
Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg
1205 1210 1215
Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val
1220 1225 1230
Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys
1235 1240 1245
Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly
1250 1255 1260
Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu
1265 1270 1275
Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu
1280 1285 1290
Phe Val Gln Asn Arg Asn Asn Ser Gly Gly Ser Pro Lys Lys Lys
1295 1300 1305
Arg Lys Val Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1310 1315 1320
<210> 23
<211> 1252
<212> PRT
<213> Artificial Sequence
<220>
<223> LBCPF1-2NLS
<400> 23
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Glu Thr Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg
165 170 175
Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe
180 185 190
Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys
195 200 205
Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly
210 215 220
Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn
225 230 235 240
Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly
245 250 255
Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu
260 265 270
Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser
275 280 285
Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu
290 295 300
Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile
305 310 315 320
Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala
325 330 335
Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp
340 345 350
Ile Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr
355 360 365
Asp Asp Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu
370 375 380
Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu
385 390 395 400
Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu
405 410 415
Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly
420 425 430
Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu
435 440 445
Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser
450 455 460
Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys
465 470 475 480
Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr
485 490 495
Asp Ile Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr
500 505 510
Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln
515 520 525
Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr
530 535 540
Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met
545 550 555 560
Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val
565 570 575
Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn
580 585 590
Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr
595 600 605
Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys
610 615 620
Lys Gly Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe
625 630 635 640
Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp
645 650 655
Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr
660 665 670
Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser
675 680 685
Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe
690 695 700
Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn
705 710 715 720
Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly
725 730 735
Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser
740 745 750
Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala
755 760 765
Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp
770 775 780
Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile
785 790 795 800
Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr
805 810 815
Glu Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly
820 825 830
Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly
835 840 845
Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn
850 855 860
Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys
865 870 875 880
Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu
885 890 895
Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys
900 905 910
Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp
915 920 925
Leu Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val
930 935 940
Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val
945 950 955 960
Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr
965 970 975
Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn
980 985 990
Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro
995 1000 1005
Ser Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile
1010 1015 1020
Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr
1025 1030 1035
Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn
1040 1045 1050
Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr
1055 1060 1065
Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn
1070 1075 1080
Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys
1085 1090 1095
Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile
1100 1105 1110
Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser
1115 1120 1125
Phe Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile
1130 1135 1140
Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn
1145 1150 1155
Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu
1160 1165 1170
Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn
1175 1180 1185
Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala
1190 1195 1200
Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys
1205 1210 1215
Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Ser Gly Gly
1220 1225 1230
Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro Lys Lys
1235 1240 1245
Lys Arg Lys Val
1250
<210> 24
<211> 1664
<212> PRT
<213> Artificial Sequence
<220>
<223> dFNCPF1-PBE-2NLS
<400> 24
Met Pro Lys Lys Lys Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala
1 5 10 15
Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val
20 25 30
Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu
35 40 45
Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn
50 55 60
Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu
65 70 75 80
Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser
85 90 95
Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser
100 105 110
Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His
115 120 125
His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser
130 135 140
Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp
145 150 155 160
Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg
165 170 175
Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile
180 185 190
Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro
195 200 205
Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg
210 215 220
Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser Glu
225 230 235 240
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Leu Lys Ser Ile Tyr
245 250 255
Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Glu
260 265 270
Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly Leu
275 280 285
Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys Gln
290 295 300
Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser Ser
305 310 315 320
Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr Phe
325 330 335
Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys Ser
340 345 350
Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp Ser
355 360 365
Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys Lys
370 375 380
Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp Asn
385 390 395 400
Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp Glu
405 410 415
Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe Lys
420 425 430
Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile Pro
435 440 445
Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe Leu
450 455 460
Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu Ala
465 470 475 480
Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr Phe
485 490 495
Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser Leu
500 505 510
Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln Ser
515 520 525
Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn Gly
530 535 540
Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr Ser
545 550 555 560
Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val Leu
565 570 575
Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile Asp
580 585 590
Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe Tyr
595 600 605
Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys Glu
610 615 620
Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp Leu
625 630 635 640
Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser Gln
645 650 655
Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu Tyr
660 665 670
Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys Lys
675 680 685
Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu Ser
690 695 700
Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg Asp
705 710 715 720
Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala Ala
725 730 735
Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu Ala
740 745 750
Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu Gln
755 760 765
Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp Gln
770 775 780
Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln Ser
785 790 795 800
Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu Val
805 810 815
Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr Asn
820 825 830
Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys Phe
835 840 845
Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys Asn
850 855 860
Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys Tyr
865 870 875 880
Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp Lys
885 890 895
Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr Lys
900 905 910
Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser Ala
915 920 925
Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile Arg
930 935 940
Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr Glu
945 950 955 960
Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe Tyr
965 970 975
Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe Arg
980 985 990
Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg Glu
995 1000 1005
Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu
1010 1015 1020
Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe
1025 1030 1035
Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro
1040 1045 1050
Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn
1055 1060 1065
Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe
1070 1075 1080
Tyr Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys
1085 1090 1095
Glu Ala Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser
1100 1105 1110
Val Phe Glu Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp
1115 1120 1125
Lys Phe Phe Phe His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser
1130 1135 1140
Gly Ala Asn Lys Phe Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu
1145 1150 1155
Lys Ala Asn Asp Val His Ile Leu Ser Ile Ala Arg Gly Glu Arg
1160 1165 1170
His Leu Ala Tyr Tyr Thr Leu Val Asp Gly Lys Gly Asn Ile Ile
1175 1180 1185
Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp Arg Met Lys Thr
1190 1195 1200
Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp Arg Asp Ser
1205 1210 1215
Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu Met Lys
1220 1225 1230
Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu Val
1235 1240 1245
Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly
1250 1255 1260
Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys
1265 1270 1275
Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys
1280 1285 1290
Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln
1295 1300 1305
Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr
1310 1315 1320
Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys
1325 1330 1335
Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser
1340 1345 1350
Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys
1355 1360 1365
Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys
1370 1375 1380
Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser
1385 1390 1395
Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp Lys Asn His
1400 1405 1410
Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu Leu Glu Lys
1415 1420 1425
Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly Glu Cys Ile
1430 1435 1440
Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe Phe Ala Lys
1445 1450 1455
Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn Ser Lys
1460 1465 1470
Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp Val
1475 1480 1485
Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro
1490 1495 1500
Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly
1505 1510 1515
Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys
1520 1525 1530
Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln
1535 1540 1545
Asn Arg Asn Asn Thr Arg Asp Ser Gly Gly Ser Thr Asn Leu Ser
1550 1555 1560
Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu
1565 1570 1575
Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn
1580 1585 1590
Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser
1595 1600 1605
Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
1610 1615 1620
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys
1625 1630 1635
Ile Lys Met Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1640 1645 1650
Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1655 1660
<210> 25
<211> 1592
<212> PRT
<213> Artificial Sequence
<220>
<223> dLBCPF1-PBE-2NLS
<400> 25
Met Pro Lys Lys Lys Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala
1 5 10 15
Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val
20 25 30
Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu
35 40 45
Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn
50 55 60
Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu
65 70 75 80
Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser
85 90 95
Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser
100 105 110
Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His
115 120 125
His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser
130 135 140
Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp
145 150 155 160
Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg
165 170 175
Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile
180 185 190
Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro
195 200 205
Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg
210 215 220
Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser Glu
225 230 235 240
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Leu Lys Ser Lys Leu
245 250 255
Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys
260 265 270
Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu
275 280 285
Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys
290 295 300
Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser
305 310 315 320
Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys
325 330 335
Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn
340 345 350
Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys
355 360 365
Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu
370 375 380
Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr
385 390 395 400
Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu
405 410 415
Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu
420 425 430
Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile
435 440 445
Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser
450 455 460
Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val
465 470 475 480
Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe
485 490 495
Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn
500 505 510
Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu
515 520 525
Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu
530 535 540
Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu
545 550 555 560
Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu
565 570 575
Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn
580 585 590
Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn
595 600 605
Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys
610 615 620
Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser
625 630 635 640
Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala
645 650 655
Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln
660 665 670
Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe
675 680 685
Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val
690 695 700
Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn
705 710 715 720
Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu
725 730 735
Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val
740 745 750
Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr
755 760 765
Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly
770 775 780
Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg
785 790 795 800
Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys
805 810 815
Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys
820 825 830
Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val
835 840 845
Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile
850 855 860
Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn
865 870 875 880
Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser
885 890 895
Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr
900 905 910
Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln
915 920 925
Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys
930 935 940
Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp
945 950 955 960
Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe
965 970 975
Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly
980 985 990
Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu
995 1000 1005
Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp
1010 1015 1020
Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp
1025 1030 1035
Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala
1040 1045 1050
Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
1055 1060 1065
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile
1070 1075 1080
Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly
1085 1090 1095
Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn
1100 1105 1110
Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu
1115 1120 1125
Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr
1130 1135 1140
Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln
1145 1150 1155
Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val
1160 1165 1170
Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val
1175 1180 1185
Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile
1190 1195 1200
Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala
1205 1210 1215
Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu
1220 1225 1230
Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile
1235 1240 1245
Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val
1250 1255 1260
Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys
1265 1270 1275
Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp
1280 1285 1290
Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp
1295 1300 1305
Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg
1310 1315 1320
Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp
1325 1330 1335
Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys
1340 1345 1350
Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys
1355 1360 1365
Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met
1370 1375 1380
Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg Thr Asp
1385 1390 1395
Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile Phe
1400 1405 1410
Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro
1415 1420 1425
Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val
1430 1435 1440
Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu
1445 1450 1455
Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr
1460 1465 1470
Ala Gln Thr Ser Val Lys His Thr Arg Asp Ser Gly Gly Ser Thr
1475 1480 1485
Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
1490 1495 1500
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val
1505 1510 1515
Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr
1520 1525 1530
Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala
1535 1540 1545
Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly
1550 1555 1560
Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Pro Lys Lys Lys
1565 1570 1575
Arg Lys Val Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1580 1585 1590
<210> 26
<211> 1731
<212> PRT
<213> Artificial Sequence
<220>
<223> dFNCPF1-ABE7.10-2NLS
<400> 26
Met Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Ser Glu Val Glu
1 5 10 15
Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg
20 25 30
Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn
35 40 45
Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp
50 55 60
Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val
65 70 75 80
Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu
85 90 95
Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg
100 105 110
Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu
115 120 125
Met Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr
130 135 140
Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe
145 150 155 160
Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser
165 170 175
Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro
180 185 190
Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
195 200 205
Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala
210 215 220
Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly
225 230 235 240
Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg
245 250 255
Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu
260 265 270
Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr
275 280 285
Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile
290 295 300
His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr
305 310 315 320
Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn
325 330 335
His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala
340 345 350
Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln
355 360 365
Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser
370 375 380
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
385 390 395 400
Ser Gly Gly Ser Ser Gly Gly Ser Leu Lys Ser Ile Tyr Gln Glu Phe
405 410 415
Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Glu Leu Ile Pro
420 425 430
Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly Leu Ile Leu Asp
435 440 445
Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys Gln Ile Ile Asp
450 455 460
Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser Ser Val Cys Ile
465 470 475 480
Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr Phe Lys Leu Lys
485 490 495
Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys Ser Ala Lys Asp
500 505 510
Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp Ser Glu Lys Phe
515 520 525
Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys Lys Gly Gln Glu
530 535 540
Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp Asn Gly Ile Glu
545 550 555 560
Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp Glu Ala Leu Glu
565 570 575
Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe Lys Gly Phe His
580 585 590
Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile Pro Thr Ser Ile
595 600 605
Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe Leu Glu Asn Lys
610 615 620
Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu Ala Ile Asn Tyr
625 630 635 640
Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr Phe Asp Ile Asp
645 650 655
Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser Leu Asp Glu Val
660 665 670
Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln Ser Gly Ile Thr
675 680 685
Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn Gly Glu Asn Thr
690 695 700
Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr Ser Gln Gln Ile
705 710 715 720
Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val Leu Phe Lys Gln
725 730 735
Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile Asp Lys Leu Glu
740 745 750
Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe Tyr Glu Gln Ile
755 760 765
Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys Glu Thr Leu Ser
770 775 780
Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp Leu Ser Lys Ile
785 790 795 800
Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser Gln Gln Val Phe
805 810 815
Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu Tyr Ile Thr Gln
820 825 830
Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys Lys Glu Gln Glu
835 840 845
Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu Ser Leu Glu Thr
850 855 860
Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg Asp Ile Asp Lys
865 870 875 880
Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala Ala Ile Pro Met
885 890 895
Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu Ala Gln Ile Ser
900 905 910
Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu Gln Ala Ser Ala
915 920 925
Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp Gln Thr Asn Asn
930 935 940
Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln Ser Glu Asp Lys
945 950 955 960
Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu Val Phe Glu Glu
965 970 975
Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr Asn Lys Ile Arg
980 985 990
Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys Phe Lys Leu Asn
995 1000 1005
Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys Asn Lys Glu
1010 1015 1020
Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys Tyr Tyr
1025 1030 1035
Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp Lys
1040 1045 1050
Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr
1055 1060 1065
Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe
1070 1075 1080
Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu
1085 1090 1095
Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln
1100 1105 1110
Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys
1115 1120 1125
Phe Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp
1130 1135 1140
Lys Asp Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser
1145 1150 1155
Ile Asp Glu Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu
1160 1165 1170
Thr Phe Glu Asn Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn
1175 1180 1185
Gln Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser
1190 1195 1200
Ala Tyr Ser Lys Gly Arg Pro Asn Leu His Thr Leu Tyr Trp Lys
1205 1210 1215
Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp Val Val Tyr Lys Leu
1220 1225 1230
Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln Ser Ile Pro Lys
1235 1240 1245
Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn Lys Asn Lys
1250 1255 1260
Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu Ile Lys
1265 1270 1275
Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro Ile
1280 1285 1290
Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu
1295 1300 1305
Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu
1310 1315 1320
Ser Ile Ala Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val
1325 1330 1335
Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile
1340 1345 1350
Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala
1355 1360 1365
Ile Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile
1370 1375 1380
Asn Asn Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val
1385 1390 1395
His Glu Ile Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val
1400 1405 1410
Phe Glu Asp Leu Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val
1415 1420 1425
Glu Lys Gln Val Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys
1430 1435 1440
Leu Asn Tyr Leu Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly
1445 1450 1455
Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe
1460 1465 1470
Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala
1475 1480 1485
Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln
1490 1495 1500
Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe
1505 1510 1515
Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe
1520 1525 1530
Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys
1535 1540 1545
Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe
1550 1555 1560
Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr
1565 1570 1575
Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu
1580 1585 1590
Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser
1595 1600 1605
Asp Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile
1610 1615 1620
Leu Gln Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu
1625 1630 1635
Ile Ser Pro Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg
1640 1645 1650
Gln Ala Pro Lys Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala
1655 1660 1665
Tyr His Ile Gly Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys
1670 1675 1680
Asn Asn Gln Glu Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu
1685 1690 1695
Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn Asn Ser Gly Gly Ser
1700 1705 1710
Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro Lys Lys Lys
1715 1720 1725
Arg Lys Val
1730
<210> 27
<211> 1659
<212> PRT
<213> Artificial Sequence
<220>
<223> dLBCPF1-ABE7.10-2NLS
<400> 27
Met Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Ser Glu Val Glu
1 5 10 15
Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg
20 25 30
Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn
35 40 45
Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp
50 55 60
Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val
65 70 75 80
Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu
85 90 95
Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg
100 105 110
Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu
115 120 125
Met Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr
130 135 140
Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe
145 150 155 160
Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser
165 170 175
Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro
180 185 190
Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
195 200 205
Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala
210 215 220
Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly
225 230 235 240
Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg
245 250 255
Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu
260 265 270
Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr
275 280 285
Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile
290 295 300
His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr
305 310 315 320
Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn
325 330 335
His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala
340 345 350
Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln
355 360 365
Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser
370 375 380
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
385 390 395 400
Ser Gly Gly Ser Ser Gly Gly Ser Leu Lys Ser Lys Leu Glu Lys Phe
405 410 415
Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro
420 425 430
Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu
435 440 445
Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp
450 455 460
Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu
465 470 475 480
Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr
485 490 495
Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys
500 505 510
Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe
515 520 525
Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys
530 535 540
Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe
545 550 555 560
Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys
565 570 575
Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr
580 585 590
Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys
595 600 605
His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp
610 615 620
Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln
625 630 635 640
Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu
645 650 655
Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn
660 665 670
Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln
675 680 685
Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr
690 695 700
Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn
705 710 715 720
Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn
725 730 735
Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala
740 745 750
Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg
755 760 765
Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala
770 775 780
Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys
785 790 795 800
Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp
805 810 815
Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp
820 825 830
Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp
835 840 845
Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile
850 855 860
Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys
865 870 875 880
Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr
885 890 895
Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile
900 905 910
Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp
915 920 925
Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp
930 935 940
Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser
945 950 955 960
Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln
965 970 975
Lys Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr
980 985 990
Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser
995 1000 1005
Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys
1010 1015 1020
Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu
1025 1030 1035
Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser
1040 1045 1050
Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu
1055 1060 1065
Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu
1070 1075 1080
Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu
1085 1090 1095
Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
1100 1105 1110
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu
1115 1120 1125
His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly
1130 1135 1140
Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala
1145 1150 1155
Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro
1160 1165 1170
Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu
1175 1180 1185
Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr
1190 1195 1200
Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile
1205 1210 1215
Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp
1220 1225 1230
Asn Pro Tyr Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Leu
1235 1240 1245
Tyr Ile Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr
1250 1255 1260
Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys
1265 1270 1275
Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe
1280 1285 1290
Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu
1295 1300 1305
Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu Leu
1310 1315 1320
Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser
1325 1330 1335
Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
1340 1345 1350
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp
1355 1360 1365
Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr
1370 1375 1380
Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln
1385 1390 1395
Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile
1400 1405 1410
Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr
1415 1420 1425
Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile
1430 1435 1440
Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr
1445 1450 1455
Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys
1460 1465 1470
Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys
1475 1480 1485
Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala
1490 1495 1500
Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly
1505 1510 1515
Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr
1520 1525 1530
Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn
1535 1540 1545
Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val
1550 1555 1560
Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala
1565 1570 1575
Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala
1580 1585 1590
Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys
1595 1600 1605
Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser
1610 1615 1620
Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Ser
1625 1630 1635
Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro
1640 1645 1650
Lys Lys Lys Arg Lys Val
1655
<210> 28
<211> 1656
<212> PRT
<213> Artificial Sequence
<220>
<223> LBCPF1-ABE2-X2
<400> 28
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile
165 170 175
Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys
180 185 190
Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys
195 200 205
Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe
210 215 220
Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile
225 230 235 240
Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn
245 250 255
Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys
260 265 270
Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser
275 280 285
Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe
290 295 300
Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys
305 310 315 320
Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile
325 330 335
Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe
340 345 350
Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp
355 360 365
Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp
370 375 380
Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu
385 390 395 400
Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu
405 410 415
Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser
420 425 430
Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys
435 440 445
Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys
450 455 460
Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr
465 470 475 480
Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
485 490 495
Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr
500 505 510
Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Ala
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Leu Met Pro Lys
1220 1225 1230
Lys Lys Arg Lys Val Ser Gly Gly Ser Ser Glu Val Glu Phe Ser
1235 1240 1245
His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala
1250 1255 1260
Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn
1265 1270 1275
Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His
1280 1285 1290
Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly
1295 1300 1305
Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val
1310 1315 1320
Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
1325 1330 1335
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly
1340 1345 1350
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn
1355 1360 1365
His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
1370 1375 1380
Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys
1385 1390 1395
Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser
1400 1405 1410
Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala
1415 1420 1425
Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu Val
1430 1435 1440
Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala
1445 1450 1455
Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu
1460 1465 1470
Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
1475 1480 1485
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
1490 1495 1500
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr
1505 1510 1515
Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
1520 1525 1530
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala
1535 1540 1545
Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro
1550 1555 1560
Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp
1565 1570 1575
Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln
1580 1585 1590
Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly
1595 1600 1605
Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser
1610 1615 1620
Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser
1625 1630 1635
Leu Lys Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys
1640 1645 1650
Lys Lys Lys
1655
<210> 29
<211> 1663
<212> PRT
<213> Artificial Sequence
<220>
<223> LBCPF1-ABE2-X3
<400> 29
Met Pro Lys Lys Lys Arg Lys Val Ser Lys Leu Glu Lys Phe Thr Asn
1 5 10 15
Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val Gly
20 25 30
Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp Glu
35 40 45
Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr
50 55 60
Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys Asn
65 70 75 80
Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys
85 90 95
Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile
100 105 110
Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys
115 120 125
Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu
130 135 140
Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly
145 150 155 160
Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser Thr
165 170 175
Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser
180 185 190
Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu
195 200 205
Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu
210 215 220
Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly
225 230 235 240
Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly
245 250 255
Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys
260 265 270
Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu
275 280 285
Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp
290 295 300
Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu
305 310 315 320
Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp
325 330 335
Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser
340 345 350
Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg Asp Lys
355 360 365
Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala Val Val
370 375 380
Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly
385 390 395 400
Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser
405 410 415
Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile
420 425 430
Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe Val
435 440 445
Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys
450 455 460
Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe
465 470 475 480
Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp
485 490 495
Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile Tyr Asp
500 505 510
Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe
515 520 525
Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp
530 535 540
Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr
545 550 555 560
Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile
565 570 575
Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu
580 585 590
Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys
595 600 605
Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys
610 615 620
Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp Cys His
625 630 635 640
Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp
645 650 655
Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp
660 665 670
Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser
675 680 685
Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly
690 695 700
Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser
705 710 715 720
His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp
725 730 735
Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe
740 745 750
Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala
755 760 765
Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr
770 775 780
Thr Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln
785 790 795 800
Tyr Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile
805 810 815
Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp Asn
820 825 830
Pro Tyr Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile
835 840 845
Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn
850 855 860
Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His
865 870 875 880
Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn
885 890 895
Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser
900 905 910
Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val
915 920 925
Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val Lys
930 935 940
Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys
945 950 955 960
Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly
965 970 975
Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser
980 985 990
Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr
995 1000 1005
Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr
1010 1015 1020
Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe
1025 1030 1035
Asp Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala
1040 1045 1050
Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys
1055 1060 1065
Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg
1070 1075 1080
Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu
1085 1090 1095
Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr
1100 1105 1110
Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys
1115 1120 1125
Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln
1130 1135 1140
Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile
1145 1150 1155
Ser Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn
1160 1165 1170
Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala
1175 1180 1185
Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly
1190 1195 1200
Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile
1205 1210 1215
Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val
1220 1225 1230
Lys His Lys Leu Met Pro Lys Lys Lys Arg Lys Val Ser Gly Gly
1235 1240 1245
Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala
1250 1255 1260
Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val
1265 1270 1275
Gly Ala Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp
1280 1285 1290
Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile
1295 1300 1305
Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu
1310 1315 1320
Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met Cys
1325 1330 1335
Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly
1340 1345 1350
Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val
1355 1360 1365
Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly
1370 1375 1380
Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg
1385 1390 1395
Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser
1400 1405 1410
Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr
1415 1420 1425
Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser
1430 1435 1440
Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met
1445 1450 1455
Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu
1460 1465 1470
Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly
1475 1480 1485
Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His
1490 1495 1500
Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn
1505 1510 1515
Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys
1520 1525 1530
Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val
1535 1540 1545
Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu
1550 1555 1560
Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu Ile
1565 1570 1575
Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr
1580 1585 1590
Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala
1595 1600 1605
Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
1610 1615 1620
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
1625 1630 1635
Gly Gly Ser Ser Gly Gly Ser Leu Lys Lys Arg Pro Ala Ala Thr
1640 1645 1650
Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1655 1660
<210> 30
<211> 1526
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 30
ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60
agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120
tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180
tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240
gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300
ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360
gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420
agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480
taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540
aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600
tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 660
cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 720
ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 780
gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 840
caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900
ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960
tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020
taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080
atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140
cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200
ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 1260
gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1320
cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1380
acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1440
ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1500
tctgcaggtc gaagcttgaa gcaaac 1526

Claims (20)

1. a kind of system for carrying out base editor to the target sequence in organism genome, it includes it is following i) in v) extremely One item missing:
I) base editor fusion protein and guide RNA；
Ii) the expression construct and guide RNA of the nucleotide sequence comprising encoding base editor fusion protein；
Iii) base editor fusion protein, and the expression construct of the nucleotide sequence comprising encoding guide RNA；
Iv) the expression construct of the nucleotide sequence comprising encoding base editor fusion protein, and the core comprising encoding guide RNA The expression construct of nucleotide sequence；
V) the expression building of the nucleotide sequence of the nucleotide sequence comprising encoding base editor fusion protein and coding guide RNA Body；
Wherein the base editor fusion protein includes the Cpf1 and deaminase of DNA cleavage activity missing, and the guide RNA can By the target sequence in the base editor fusion protein target gene group, lead to one or more C to T or A in the target sequence To the substitution of G.
2. the system of claim 1, wherein the Cpf1 of DNA cleavage activity missing is the FnCpf1 of DNA cleavage activity missing, Such as the FnCpf1 of the DNA cleavage activity missing is mutated relative to wild type FnCpf1 comprising D917A.
3. the system of claim 1, wherein the Cpf1 of DNA cleavage activity missing is the AsCpf1 of DNA cleavage activity missing, Such as the AsCpf1 of the DNA cleavage activity missing is mutated relative to wild type AsCpf1 comprising D908A.
4. the system of claim 1, wherein the Cpf1 of DNA cleavage activity missing is the LbCpf1 of DNA cleavage activity missing, Such as the LbCpf1 of the DNA cleavage activity missing is mutated relative to wild type LbCpf1 comprising D832A.
5. the system of claim 1, wherein the deaminase is cytidine deaminase, such as apolipoprotein B mRNA edits complex (APOBEC) family's deaminase.
6. the system of claim 5, wherein the cytidine deaminase is the cytidine deaminase of APOBEC1 deaminase or activation-inducing (AID)。
7. the system of claim 5, wherein the base editor fusion protein also includes uracil dna glycosylase inhibitor (UGI)。
8. the system of claim 1, wherein the deaminase is DNA dependent form adenine deaminase, preferably single stranded DNA dependent form Adenine deaminase.
9. the system of claim 8, wherein the DNA dependent form adenine deaminase is Escherichia coli tRNA adenine deaminase The variant of TadA (ecTadA) can especially receive variant of the single stranded DNA as substrate.
10. the system of claim 9, wherein the DNA dependent form adenine deaminase includes one relative to wild type ecTadA Or multiple groups mutation selected from the following:
1) A106V and D108N；
2) D147Y and E155V；
3) L84F, H123Y and I156F；
4)A142N；
5) H36L, R51L, S146C and K157N；
6)P48S/T/A；
7)A142N；
8)W23L/R；With
9)R152H/P。
11. the system of claim 10, wherein the DNA dependent form adenine deaminase relative to wild type ecTadA include with Lower mutation: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, D147Y, E155V and R152P.
12. the N-terminal fusion of the system of claim 9, the DNA dependent form adenine deaminase has corresponding wild type adenine The N-terminal of deaminase, the preferably described DNA dependent form adenine deaminase passes through connector and corresponding wild type adenine deaminase Fusion.
13. the system of claim 1, wherein the deaminase is fused the end N of the Cpf1 lacked to the DNA cleavage activity End or in which the deaminase are fused the C-terminal of the Cpf1 lacked to DNA cleavage activity.
14. the system of claim 1, wherein the deaminase and the Cpf1 of DNA cleavage activity missing are merged by connector.
15. the system of claim 1, wherein the base editor fusion protein includes also nuclear location sequence in its N-terminal and/or C-terminal It arranges (NLS).
16. the system of claim 1, wherein the nucleotide sequence of the encoding base editor fusion protein is directed to pending base The organism of editor carries out codon optimization.
17. the system of claim 1, the nucleotide sequence of the encoding base editor fusion protein and/or the coding guide The nucleotide sequence of RNA is operably connected with expression regulation element.
18. the system of claim 17, wherein the controlling element is promoter, such as 35S promoter, corn Ubi-1 starting Son, wheat U6 promoter, rice U3 promoter or corn U3 promoter.
19. a kind of method for generating genetically modified organism, including by the system introducing of any one of claim 1-18 Biological cell, thus the base editor fusion protein is targeted the target sequence in the cellular genome by the guide RNA, Lead to the substitution of one or more C to T or A to G in the target sequence.
20. the method for claim 19, wherein the organism be selected from mammal for example people, mouse, rat, monkey, dog, pig, sheep, Ox, cat；Poultry such as chicken, duck, goose；Plant, including monocotyledon and dicotyledon, for example, rice, corn, wheat, sorghum, Barley, soybean, peanut, arabidopsis.
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