CN112430597A

CN112430597A - CasRx preparation for silencing target gene and application thereof

Info

Publication number: CN112430597A
Application number: CN202011328426.1A
Authority: CN
Inventors: 张苗苗; 洪丹; 胡迅
Original assignee: Shenzhen Ruiji Biotechnology Co ltd
Current assignee: Shenzhen Ruiji Biotechnology Co ltd
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-03-02
Also published as: WO2022110260A1

Abstract

The invention provides a CasRx preparation for silencing a target gene and application, belonging to the technical field of genetic engineering, wherein the CasRx preparation comprises CasRx mRNA and sgRNA; the CasRx mRNA encodes a CasRx protein, and the amino acid sequence of the CasRx protein is shown as SEQ ID No. 1; the sgRNA is set according to a target gene. The CasRx preparation provided by the invention is simple, rapid and high in specificity, and can be used for rapidly, efficiently and highly specifically knocking down the expression level of the target gene and interfering RNA.

Description

CasRx preparation for silencing target gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a CasRx preparation for silencing a target gene and application thereof.

Background

In recent years, CRISPR/Cas9 technology has received much attention due to its powerful and convenient DNA editing capabilities. In 2016, the Zhang Pioneng laboratory discovered a new Cas protein Cas13a that could target RNA for cleavage. Then, Cas13b and Cas13c targeting RNA are discovered successively. Due to the characteristics of the targeting RNA of the Cas13 family protein, the protein has unique advantages in detection and treatment of certain specific diseases in theory, and thus becomes a research hotspot in recent years. In 2018, Patrick Hsu laboratories, university of california, berkeley, discovered the Cas13d family. They found that Cas13 d-mediated gene silencing was more specific (Cas 13d did not miss as compared to hundreds of shRNA misses) and knockout efficiency (shRNA up to 65%, Cas13d up to 96%) than RNA interference techniques. Compared with a Cas 9-mediated gene knockout technology, Cas13 d-mediated gene silencing does not change genome DNA, so that the gene silencing is reversible, and the treatment of certain acquired diseases (such as acquired metabolic diseases such as hyperlipidemia caused by poor living habits) is more advantageous. Among them, CasRx proteins of Cas13d family are considered to be the most advantageous Cas13 protein in future applications due to their small size and high efficiency.

Currently, the conventional approach to gene silencing using the CasRx system is to package both CasRx-expressing and sgRNA directed against a target gene in AAV, followed by transfection and gene silencing of the target cells. The disadvantage of this method is that AAV virus infection is extensive and lacks targeting, which causes the CasRx system to function in normal tissues other than target organs and target cells and causes adverse reactions. Meanwhile, the adenovirus vector can stimulate antigen presenting cells of an organism to generate specific immunoreaction to isohexide antigen while being combined with target cells of the organism or promote the natural immunity of the organism to the adenovirus vector, which can influence the tolerance of drugs, thereby influencing the gene silencing and the treatment effect.

Disclosure of Invention

In view of the above, the present invention provides a CasRx preparation for silencing a target gene and an application thereof, wherein CasRx mRNA and sgRNA specific to the target gene are directly introduced into a target cell, so that a gap of a CasRx-based efficient targeted gene silencing system in the mRNA preparation field is filled up, and the above-mentioned defects of an AAV virus-carried CasRx system are overcome.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a CasRx preparation for silencing a target gene, which comprises CasRx mRNA and sgRNA;

the CasRx mRNA encodes a CasRx protein, and the amino acid sequence of the CasRx protein is shown as SEQ ID No. 1;

the sgRNA is set according to a target gene;

the mass ratio of CasRx mRNA to sgRNA is 5-100: 1.

Preferably, the CasRx mRNA further comprises a nucleotide sequence encoding a nuclear localization signal peptide.

Preferably, the nucleotide sequence encoding the nuclear localization signal peptide is shown as SEQ ID No. 2.

Preferably, the CasRx mRNA is from any one of SEQ ID Nos. 3-18.

Preferably, when the target gene is Pcsk 9gene, the nucleotide sequence of the sgRNA is shown as SEQ ID No.19 and/or SEQ ID No. 20.

Preferably, when the target gene is the Vegfa gene, the nucleotide sequence of the sgRNA is shown as SEQ ID No.21 and/or SEQ ID No. 22.

Preferably, when the target gene is TYR gene, the nucleotide sequence of the sgRNA is shown as SEQ ID No.23 and/or SEQ ID No. 24.

The invention also provides application of the CasRx preparation in gene silencing and/or RNA interference.

The invention provides a CasRx preparation for silencing a target gene and application, wherein the CasRx preparation comprises CasRx mRNA and sgRNA; the CasRx mRNA encodes a CasRx protein, and the amino acid sequence of the CasRx protein is shown as SEQ ID No. 1; the sgRNA is set according to a target gene; the mass ratio of CasRx mRNA to sgRNA is 5-100: 1.

Drawings

FIG. 1 is a structural diagram of CasRx mRNA;

FIG. 2A shows the result of the cell-level downregulation of Pcsk 9gene expression by CasRx mRNA, which was transfected into mouse liver primary cells with 10 sgRNAs, and the result showed that sgRNA6 and sgRNA7 had significant downregulation effect on Pcsk9, and the effect was the best when the two were combined, and Pcsk9 mRNA level was only 12% of that of the control group; fig. 2B shows alignment of the CasRx sequences for 16 different species optimization schemes for this optimal combination of sgRNA6 and sgRNA7, seq 11 having the best editing effect, seq10 times.

FIG. 3 shows the result that CasRx mRNA can down-regulate the expression of Pcsk 9gene in mouse, and when CasRx encoding mRNA and Pcsk9 sgRNA6 and 7 are coated with liposome and injected into mouse, the level of Pcsk9 mRNA in mouse liver is greatly reduced and the expression level of PCSK9 protein in serum is obviously reduced;

FIG. 4 shows the results of the cholesterol levels in the blood of mice injected with CasRx mRNA and sgRNA, which resulted in a nearly 60% decrease in the total cholesterol concentration in the blood after the expression level of PCSK9 was down-regulated using the CasRx editing system;

FIG. 5A shows the results of CasRx mRNA downregulating the expression of Vegfa gene at the cellular level, and when CasRx mRNA and 10 sgRNAs were transfected into N2a cells, sgRNA5 and sgRNA8 showed significant downregulation of Vegfa mRNA level, which was only 22% of that of the control group when they were combined; fig. 5B shows alignment of the CasRx sequences for 16 different species optimization schemes for this optimal combination of sgRNA5 and sgRNA8, seq 11 having the best editing effect, seq10 times.

FIG. 6 shows the result that CasRx mRNA down-regulates the expression of Vegfa gene in a mouse, the mRNA coding for CasRx and Vegfa sgRNA5 and 8 are coated by liposome and then injected into the fundus oculi of the mouse together, the Vegfa mRNA level in the fundus oculi of the mouse is greatly reduced, and the expression level of VEGFA protein in the tissues of the eye is obviously reduced;

FIG. 7 shows the eye function results of mice injected with CasRx mRNA and sgRNA to the fundus, before the expression level of VEGFA is down-regulated by the CasRx editing system, the macular mouse is insensitive to light, and after the CasRx mRNA editing system is adopted, the macular mouse is in the dark for a long time to restore the sensitivity to light to the normal level;

FIG. 8A shows the result of the down-regulation of TYR gene expression at cellular level by CasRx mRNA, transfection of mouse melanocytes with CasRx mRNA and 10 sgRNAs, sgRNA3 and sgRNA4, which had significant down-regulation of TYR mRNA levels, which were only 16% of the control group when they were combined; fig. 8B shows alignment of the CasRx sequences for 16 different species optimization schemes for this optimal combination of sgRNA3 and sgRNA4, seq 11 having the best editing effect, seq10 times.

FIG. 9 shows the result of CasRx mRNA down-regulating the expression of TYR gene in mouse, in which CasRx encoding mRNA and TYR sgRNA3 and 4 are liposome coated and injected into the skin of mouse, and the level of TYR mRNA in mouse skin is greatly reduced and the expression level of TYR protein is obviously reduced;

FIG. 10 shows the results of melanin deposition after injecting CasRx mRNA and sgRNA into the skin of mice, in which normal saline was injected into the control mice and CasRx mRNA and sgRNA were injected into the experimental mice, showing that the melanin deposition in the skin of the mice is reduced.

Detailed Description

The invention provides a CasRx preparation for silencing a target gene, which comprises CasRx mRNA and sgRNA; the CasRx mRNA encodes a CasRx protein, and the amino acid sequence of the CasRx protein is shown as SEQ ID No. 1; the sgRNA is set according to a target gene; the mass ratio of CasRx mRNA to sgRNA is 5-100: 1.

In the present invention, the CasRx mRNA includes the following features:

(a) a sequence of n number of linked nucleosides;

(b) a 5' UTR comprising at least one Kozak sequence;

(c)3'UTR；

(d) at least one 5' cap structure;

(e) contains a poly-A tail (polyAtail).

In the invention, the mRNA consists of uridine, cytidine, adenosine, guanosine and/or chemically modified nucleosides, and the structure is shown in figure 1.

In the present invention, the CasRx mRNA encodes the CasRx protein, and the CasRx enzyme white has RNA editing ability, which can help to regulate the gene expression pattern without changing the genome, and furthermore, the RNA life span is relatively short, which means that its change can be reversed, thereby avoiding a great risk in genetic engineering. Prior to the advent of CRISPR, RNAi was an ideal method of modulating gene expression. However, the CasRx enzymes have the great advantage of being more specific and that the system itself derived from the bacterium is not endogenous to mammalian cells and therefore less likely to interfere with the transcription native to the cell. In contrast, RNAi utilizes endogenous mechanisms for gene knock-out, which have a large impact on itself. However, the CRISPR-CasRx system has an important problem in practical use, i.e. the CasRx enzyme is inherently a relatively large protein and thus difficult to package into target tissues. In addition, due to the pre-existing immunity of human body to adenovirus, the efficient expression of CasRx cannot be realized by using adenovirus vector to express CasRx in vivo. In addition, adeno-associated viral loads are limited in their ability to deliver large gene fragments, and therefore CasRx systems based on viral vector dosage formulations do not fully exploit the advantage of CasRx in mRNA editing in vivo. mRNA has no size limitation on the expressed protein, so that the problem can be effectively solved by expressing CasRx by using mRNA, so that the RNA editing technology really plays a role in clinical application. The amino acid sequence of the CasRx protein is shown as SEQ ID No.1, and specifically comprises the following steps:

MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKN AGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIE KILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDN FLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLD NEYISTLNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLRE VMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVIN LRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSK LMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFAR MGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGMRNFIINNVISNKRFH YLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALT KIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQL YKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIK YSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAY INDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKN LSIEALFDRNEAAKFDKEKKKVSGNS。

in the present invention, the CasRx mRNA preferably further includes a nucleotide sequence encoding a nuclear localization signal peptide, the amino acid sequence of the nuclear localization signal peptide is not particularly limited, and the CasRx protein can be introduced into the nucleus of a cell, for example, SV40 NLS, and the nucleotide encoding SV40 NLS is shown in SEQ ID No.2, specifically as follows:

CCCAAGAAAAAGAGAAAGGTG。

in the invention, the CasRx mRNA is from any one of SEQ ID No. 3-18, and the specific steps are as follows:

SEQ ID No.3：

atgatcgaaaagaaaaagagcttcgcgaagggcatgggcgtgaagagcaccctggtgagcggcagcaaggtgtacatgaccaccttcgccgagg gcagcgacgctagactggagaagatcgtggagggcgacagcatcagatccgtgaacgagggcgaggccttcagcgccgagatggccgacaagaacgc cggctacaagatcggcaacgccaagttcagccatcccaagggctacgccgtggtggccaacaaccccctgtacaccggccccgtgcagcaagacatgctg ggcctgaaggagaccctggagaagagatacttcggcgagagcgccgacggcaacgacaacatctgcatccaagtgatccacaacatcctggacatcgag aagatcctggccgagtacatcaccaacgccgcctacgccgtgaacaacatcagcggcctggacaaggacatcatcggcttcggcaagttcagcaccgtgta cacctacgacgagttcaaagatcccgagcaccacagagccgccttcaacaacaacgacaagctgatcaacgccatcaaggctcagtatgacgagtttgata atttcctggacaaccctagactgggctacttcggccaagccttcttcagcaaggagggcagaaactacatcatcaactacggcaacgagtgctacgacatcct ggccctgctgagcggcctgagacactgggtggtgcacaacaacgaggaggagagcagaatcagcagaacctggctgtacaacctggacaagaacctgg acaacgagtacatcagcaccctgaactacctgtacgacagaatcaccaacgagctgaccaacagcttcagcaagaacagcgccgccaacgtgaactacatc gccgagaccctgggcatcaaccccgccgagttcgccgagcagtacttcagattcagcatcatgaaggagcagaagaacctgggcttcaacatcaccaagct gagagaggtgatgctggacagaaaggacatgagcgagatcagaaagaaccacaaggtgttcgatagcataaggaccaaggtgtacaccatgatggacttc gtgatctacagatactacatcgaggaggacgccaaggtggccgccgccaacaagagcctgcccgacaacgagaagagcctcagcgagaaggacatcttc gtgatcaacctgagaggcagcttcaacgacgatcagaaggacgcactgtattacgacgaggccaacagaatctggagaaagctggagaacatcatgcaca acatcaaggagttcagaggcaacaagacaagagagtacaagaagaaggatgcccctagactgcctagaatcctgcccgccggcagagacgtgagcgcct tcagcaagctgatgtacgccctgaccatgttcctggacggcaaggagatcaacgacctgctgaccaccctgatcaacaagtttgacaacattcagagcttcct gaaggtgatgcccctgatcggcgtgaacgccaagtttgtggaggagtacgccttcttcaaggacagcgccaagatcgccgacgagctgagactgatcaaga gtttcgcgcggatgggcgagcccatcgccgacgctagaagagccatgtacatcgacgccatcagaatcctgggcaccaacctgagctacgacgagctgaa ggccctggccgacaccttcagcctggacgagaacggcaacaagctgaagaagggcaagcacggcatgagaaacttcatcatcaacaacgtgatcagcaa caagagattccactacctgatcagatacggcgaccccgcccacctgcacgagatcgccaagaacgaggccgtggtgaagttcgtgctgggcagaatcgcc gacattcagaagaagcaagggcagaacggcaagaatcagatcgacagatactacgagacctgcatcggcaaggacaagggcaagagcgttagcgaaaa ggttgacgccctgacaaagatcatcaccggcatgaactacgatcagttcgacaagaagagaagcgtgatcgaggacaccggcagagagaacgccgagag agagaagttcaagaagatcatcagcctgtacctgaccgtgatctaccacatcctgaagaacatcgtgaacatcaacgctagatacgttatcggcttccactgcg tggagagagacgctcagctgtacaaagagaagggttacgacatcaacctgaagaagctggaggagaagggctttagcagcgtgaccaagctgtgcgccg gcatcgacgagaccgcccccgacaagagaaaggacgtggagaaggagatggccgagagagccaaggagagcatcgacagcctggagagcgccaacc ccaagctgtacgccaactacatcaagtacagcgacgagaagaaggccgaggagttcacaagacagatcaacagagagaaggccaagaccgccctgaac gcctacctgagaaacaccaagtggaacgtgatcatcagagaggacctgctgagaatcgacaacaagacctgcaccctgttcagaaacaaggccgtgcacct ggaggtggctagatacgttcatgcctacatcaacgacatcgccgaggtgaacagctactttcagctgtaccactacatcatgcagagaatcatcatgaacgag agatacgaaaagagcagcggcaaggtgagcgagtacttcgacgccgtgaacgatgagaagaaatataacgacagactgctgaagctgctgtgcgtgccct tcggctactgcatccctagattcaagaacctgagcatcgaggccctgttcgacagaaacgaggccgccaagttcgataaggaaaaaaagaaagtttcgggc aacagctaa。

SEQ ID No.4：

atgattgaaaagaagaagtcttttgctaagggaatgggagttaagtctactcttgtttctggatctaaggtttatatgactacttttgctgaaggatctgatgc tagacttgaaaagattgttgaaggagattctattagatctgttaatgaaggagaagctttttctgctgaaatggctgataagaatgctggatataagattggaaatg ctaagttttctcatcctaagggatatgctgttgttgctaataatcctctttatactggacctgttcaacaagatatgcttggacttaaggaaactcttgaaaagagatat tttggagaatctgctgatggaaatgataatatttgtattcaagttattcataatattcttgatattgaaaagattcttgctgaatatattactaatgctgcttatgctgttaat aatatttctggacttgataaggatattattggatttggaaagttttctactgtttatacttatgatgaatttaaggatcctgaacatcatagagctgcttttaataataatg ataagcttattaatgctattaaggctcaatatgatgaatttgataattttcttgataatcctagacttggatattttggacaagcttttttttctaaggaaggaagaaatta tattattaattatggaaatgaatgttatgatattcttgctcttctttctggacttagacattgggttgttcataataatgaagaagaatctagaatttctagaacttggcttt ataatcttgataagaatcttgataatgaatatatttctactcttaattatctttatgatagaattactaatgaacttactaattctttttctaagaattctgctgctaatgttaat tatattgctgaaactcttggaattaatcctgctgaatttgctgaacaatattttagattttctattatgaaggaacaaaagaatcttggatttaatattactaagcttaga gaagttatgcttgatagaaaggatatgtctgaaattagaaagaatcataaggtttttgattctattagaactaaggtttatactatgatggattttgttatttatagatatt atattgaagaagatgctaaggttgctgctgctaataagtctcttcctgataatgaaaagtctctttctgaaaaggatatttttgttattaatcttagaggatcttttaatg atgatcaaaaggatgctctttattatgatgaagctaatagaatttggagaaagcttgaaaatattatgcataatattaaggaatttagaggaaataagactagagaa tataagaagaaggatgctcctagacttcctagaattcttcctgctggaagagatgtttctgctttttctaagcttatgtatgctcttactatgtttcttgatggaaagga aattaatgatcttcttactactcttattaataagtttgataatattcaatcttttcttaaggttatgcctcttattggagttaatgctaagtttgttgaagaatatgctttttttaa ggattctgctaagattgctgatgaacttagacttattaagtcttttgctagaatgggagaacctattgctgatgctagaagagctatgtatattgatgctattagaatt cttggaactaatctttcttatgatgaacttaaggctcttgctgatactttttctcttgatgaaaatggaaataagcttaagaagggaaagcatggaatgagaaatttta ttattaataatgttatttctaataagagatttcattatcttattagatatggagatcctgctcatcttcatgaaattgctaagaatgaagctgttgttaagtttgttcttggaa gaattgctgatattcaaaagaagcaaggacaaaatggaaagaatcaaattgatagatattatgaaacttgtattggaaaggataagggaaagtctgtttctgaaa aggttgatgctcttactaagattattactggaatgaattatgatcaatttgataagaagagatctgttattgaagatactggaagagaaaatgctgaaagagaaaa gtttaagaagattatttctctttatcttactgttatttatcatattcttaagaatattgttaatattaatgctagatatgttattggatttcattgtgttgaaagagatgctcaac tttataaggaaaagggatatgatattaatcttaagaagcttgaagaaaagggattttcttctgttactaagctttgtgctggaattgatgaaactgctcctgataaga gaaaggatgttgaaaaggaaatggctgaaagagctaaggaatctattgattctcttgaatctgctaatcctaagctttatgctaattatattaagtattctgatgaaa agaaggctgaagaatttactagacaaattaatagagaaaaggctaagactgctcttaatgcttatcttagaaatactaagtggaatgttattattagagaagatctt cttagaattgataataagacttgtactctttttagaaataaggctgttcatcttgaagttgctagatatgttcatgcttatattaatgatattgctgaagttaattcttatttt caactttatcattatattatgcaaagaattattatgaatgaaagatatgaaaagtcttctggaaaggtttctgaatattttgatgctgttaatgatgaaaagaagtataat gatagacttcttaagcttctttgtgttccttttggatattgtattcctagatttaagaatctttctattgaagctctttttgatagaaatgaagctgctaagtttgataagga aaagaagaaggtttctggaaattcttga。

SEQ ID No.5：

atgattgaaaaaaaaaaatcattcgctaaaggaatgggagttaaatcaacacttgtttcaggatcaaaagtttatatgacaacattcgctgaaggatcaga tgctagacttgaaaaaattgttgaaggagattcaattagatcagttaatgaaggagaagctttctcagctgaaatggctgataaaaatgctggatataaaattgga aatgctaaattctcacatccaaaaggatatgctgttgttgctaataatccactttatacaggaccagttcaacaagatatgcttggacttaaagaaacacttgaaaa aagatatttcggagaatcagctgatggaaatgataatatttgtattcaagttattcataatattcttgatattgaaaaaattcttgctgaatatattacaaatgctgcttat gctgttaataatatttcaggacttgataaagatattattggattcggaaaattctcaacagtttatacatatgatgaattcaaagatccagaacatcatagagctgcttt caataataatgataaacttattaatgctattaaagctcaatatgatgaattcgataatttccttgataatccaagacttggatatttcggacaagctttcttctcaaaag aaggaagaaattatattattaattatggaaatgaatgttatgatattcttgctcttctttcaggacttagacattgggttgttcataataatgaagaagaatcaagaattt caagaacatggctttataatcttgataaaaatcttgataatgaatatatttcaacacttaattatctttatgatagaattacaaatgaacttacaaattcattctcaaaaa attcagctgctaatgttaattatattgctgaaacacttggaattaatccagctgaattcgctgaacaatatttcagattctcaattatgaaagaacaaaaaaatcttgg attcaatattacaaaacttagagaagttatgcttgatagaaaagatatgtcagaaattagaaaaaatcataaagttttcgattcaattagaacaaaagtttatacaat gatggatttcgttatttatagatattatattgaagaagatgctaaagttgctgctgctaataaatcacttccagataatgaaaaatcactttcagaaaaagatattttcg ttattaatcttagaggatcattcaatgatgatcaaaaagatgctctttattatgatgaagctaatagaatttggagaaaacttgaaaatattatgcataatattaaagaa ttcagaggaaataaaacaagagaatataaaaaaaaagatgctccaagacttccaagaattcttccagctggaagagatgtttcagctttctcaaaacttatgtatg ctcttacaatgttccttgatggaaaagaaattaatgatcttcttacaacacttattaataaattcgataatattcaatcattccttaaagttatgccacttattggagttaat gctaaattcgttgaagaatatgctttcttcaaagattcagctaaaattgctgatgaacttagacttattaaatcattcgctagaatgggagaaccaattgctgatgcta gaagagctatgtatattgatgctattagaattcttggaacaaatctttcatatgatgaacttaaagctcttgctgatacattctcacttgatgaaaatggaaataaactt aaaaaaggaaaacatggaatgagaaatttcattattaataatgttatttcaaataaaagattccattatcttattagatatggagatccagctcatcttcatgaaattgc taaaaatgaagctgttgttaaattcgttcttggaagaattgctgatattcaaaaaaaacaaggacaaaatggaaaaaatcaaattgatagatattatgaaacatgta ttggaaaagataaaggaaaatcagtttcagaaaaagttgatgctcttacaaaaattattacaggaatgaattatgatcaattcgataaaaaaagatcagttattgaa gatacaggaagagaaaatgctgaaagagaaaaattcaaaaaaattatttcactttatcttacagttatttatcatattcttaaaaatattgttaatattaatgctagatat gttattggattccattgtgttgaaagagatgctcaactttataaagaaaaaggatatgatattaatcttaaaaaacttgaagaaaaaggattctcatcagttacaaaa ctttgtgctggaattgatgaaacagctccagataaaagaaaagatgttgaaaaagaaatggctgaaagagctaaagaatcaattgattcacttgaatcagctaat ccaaaactttatgctaattatattaaatattcagatgaaaaaaaagctgaagaattcacaagacaaattaatagagaaaaagctaaaacagctcttaatgcttatctt agaaatacaaaatggaatgttattattagagaagatcttcttagaattgataataaaacatgtacacttttcagaaataaagctgttcatcttgaagttgctagatatgt tcatgcttatattaatgatattgctgaagttaattcatatttccaactttatcattatattatgcaaagaattattatgaatgaaagatatgaaaaatcatcaggaaaagtt tcagaatatttcgatgctgttaatgatgaaaaaaaatataatgatagacttcttaaacttctttgtgttccattcggatattgtattccaagattcaaaaatctttcaattg aagctcttttcgatagaaatgaagctgctaaattcgataaagaaaaaaaaaaagtttcaggaaattcatga。

SEQ ID No.6：

atgatcgagaagaagaagtccttcgccaagggcatgggcgtgaagtccaccctggtgtccggctccaaggtgtacatgaccaccttcgccgagggc tccgacgccaggctggagaagatcgtggagggcgactccatcaggtccgtgaacgagggcgaggccttctccgccgagatggccgacaagaacgccgg ctacaagatcggcaacgccaagttctcccaccccaagggctacgccgtggtggccaacaaccccctgtacaccggccccgtgcagcaggacatgctgggc ctgaaggagaccctggagaagaggtacttcggcgagtccgccgacggcaacgacaacatctgcatccaggtgatccacaacatcctggacatcgagaaga tcctggccgagtacatcaccaacgccgcctacgccgtgaacaacatctccggcctggacaaggacatcatcggcttcggcaagttctccaccgtgtacacct acgacgagttcaaggaccccgagcaccacagggccgccttcaacaacaacgacaagctgatcaacgccatcaaggcccagtacgacgagttcgacaactt cctggacaaccccaggctgggctacttcggccaggccttcttctccaaggagggcaggaactacatcatcaactacggcaacgagtgctacgacatcctgg ccctgctgtccggcctgaggcactgggtggtgcacaacaacgaggaggagtccaggatctccaggacctggctgtacaacctggacaagaacctggacaa cgagtacatctccaccctgaactacctgtacgacaggatcaccaacgagctgaccaactccttctccaagaactccgccgccaacgtgaactacatcgccga gaccctgggcatcaaccccgccgagttcgccgagcagtacttcaggttctccatcatgaaggagcagaagaacctgggcttcaacatcaccaagctgaggg aggtgatgctggacaggaaggacatgtccgagatcaggaagaaccacaaggtgttcgactccatcaggaccaaggtgtacaccatgatggacttcgtgatct acaggtactacatcgaggaggacgccaaggtggccgccgccaacaagtccctgcccgacaacgagaagtccctgtccgagaaggacatcttcgtgatcaa cctgaggggctccttcaacgacgaccagaaggacgccctgtactacgacgaggccaacaggatctggaggaagctggagaacatcatgcacaacatcaa ggagttcaggggcaacaagaccagggagtacaagaagaaggacgcccccaggctgcccaggatcctgcccgccggcagggacgtgtccgccttctcca agctgatgtacgccctgaccatgttcctggacggcaaggagatcaacgacctgctgaccaccctgatcaacaagttcgacaacatccagtccttcctgaaggt gatgcccctgatcggcgtgaacgccaagttcgtggaggagtacgccttcttcaaggactccgccaagatcgccgacgagctgaggctgatcaagtccttcgc caggatgggcgagcccatcgccgacgccaggagggccatgtacatcgacgccatcaggatcctgggcaccaacctgtcctacgacgagctgaaggccct ggccgacaccttctccctggacgagaacggcaacaagctgaagaagggcaagcacggcatgaggaacttcatcatcaacaacgtgatctccaacaagagg ttccactacctgatcaggtacggcgaccccgcccacctgcacgagatcgccaagaacgaggccgtggtgaagttcgtgctgggcaggatcgccgacatcc agaagaagcagggccagaacggcaagaaccagatcgacaggtactacgagacctgcatcggcaaggacaagggcaagtccgtgtccgagaaggtgga cgccctgaccaagatcatcaccggcatgaactacgaccagttcgacaagaagaggtccgtgatcgaggacaccggcagggagaacgccgagagggaga agttcaagaagatcatctccctgtacctgaccgtgatctaccacatcctgaagaacatcgtgaacatcaacgccaggtacgtgatcggcttccactgcgtggag agggacgcccagctgtacaaggagaagggctacgacatcaacctgaagaagctggaggagaagggcttctcctccgtgaccaagctgtgcgccggcatc gacgagaccgcccccgacaagaggaaggacgtggagaaggagatggccgagagggccaaggagtccatcgactccctggagtccgccaaccccaag ctgtacgccaactacatcaagtactccgacgagaagaaggccgaggagttcaccaggcagatcaacagggagaaggccaagaccgccctgaacgcctac ctgaggaacaccaagtggaacgtgatcatcagggaggacctgctgaggatcgacaacaagacctgcaccctgttcaggaacaaggccgtgcacctggag gtggccaggtacgtgcacgcctacatcaacgacatcgccgaggtgaactcctacttccagctgtaccactacatcatgcagaggatcatcatgaacgagagg tacgagaagtcctccggcaaggtgtccgagtacttcgacgccgtgaacgacgagaagaagtacaacgacaggctgctgaagctgctgtgcgtgcccttcgg ctactgcatccccaggttcaagaacctgtccatcgaggccctgttcgacaggaacgaggccgccaagttcgacaaggagaagaagaaggtgtccggcaac tcctga。

SEQ ID No.7：

atgatcgagaagaagaagagcttcgctaagggaatgggagtgaagagcacactggtgagcggaagcaaggtgtacatgacaacattcgctgaggg aagcgacgctagactggagaagatcgtggagggagacagcatcagaagcgtgaacgagggagaggctttcagcgctgagatggctgacaagaacgctg gatacaagatcggaaacgctaagttcagccaccctaagggatacgctgtggtggctaacaaccctctgtacacaggacctgtgcagcaggacatgctggga ctgaaggagacactggagaagagatacttcggagagagcgctgacggaaacgacaacatctgtatccaggtgatccacaacatcctggacatcgagaaga tcctggctgagtacatcacaaacgctgcttacgctgtgaacaacatcagcggactggacaaggacatcatcggattcggaaagttcagcacagtgtacacata cgacgagttcaaggaccctgagcaccacagagctgctttcaacaacaacgacaagctgatcaacgctatcaaggctcagtacgacgagttcgacaacttcct ggacaaccctagactgggatacttcggacaggctttcttcagcaaggagggaagaaactacatcatcaactacggaaacgagtgttacgacatcctggctctg ctgagcggactgagacactgggtggtgcacaacaacgaggaggagagcagaatcagcagaacatggctgtacaacctggacaagaacctggacaacga gtacatcagcacactgaactacctgtacgacagaatcacaaacgagctgacaaacagcttcagcaagaacagcgctgctaacgtgaactacatcgctgaga cactgggaatcaaccctgctgagttcgctgagcagtacttcagattcagcatcatgaaggagcagaagaacctgggattcaacatcacaaagctgagagagg tgatgctggacagaaaggacatgagcgagatcagaaagaaccacaaggtgttcgacagcatcagaacaaaggtgtacacaatgatggacttcgtgatctac agatactacatcgaggaggacgctaaggtggctgctgctaacaagagcctgcctgacaacgagaagagcctgagcgagaaggacatcttcgtgatcaacct gagaggaagcttcaacgacgaccagaaggacgctctgtactacgacgaggctaacagaatctggagaaagctggagaacatcatgcacaacatcaagga gttcagaggaaacaagacaagagagtacaagaagaaggacgctcctagactgcctagaatcctgcctgctggaagagacgtgagcgctttcagcaagctg atgtacgctctgacaatgttcctggacggaaaggagatcaacgacctgctgacaacactgatcaacaagttcgacaacatccagagcttcctgaaggtgatgc ctctgatcggagtgaacgctaagttcgtggaggagtacgctttcttcaaggacagcgctaagatcgctgacgagctgagactgatcaagagcttcgctagaat gggagagcctatcgctgacgctagaagagctatgtacatcgacgctatcagaatcctgggaacaaacctgagctacgacgagctgaaggctctggctgaca cattcagcctggacgagaacggaaacaagctgaagaagggaaagcacggaatgagaaacttcatcatcaacaacgtgatcagcaacaagagattccacta cctgatcagatacggagaccctgctcacctgcacgagatcgctaagaacgaggctgtggtgaagttcgtgctgggaagaatcgctgacatccagaagaagc agggacagaacggaaagaaccagatcgacagatactacgagacatgtatcggaaaggacaagggaaagagcgtgagcgagaaggtggacgctctgaca aagatcatcacaggaatgaactacgaccagttcgacaagaagagaagcgtgatcgaggacacaggaagagagaacgctgagagagagaagttcaagaa gatcatcagcctgtacctgacagtgatctaccacatcctgaagaacatcgtgaacatcaacgctagatacgtgatcggattccactgtgtggagagagacgctc agctgtacaaggagaagggatacgacatcaacctgaagaagctggaggagaagggattcagcagcgtgacaaagctgtgtgctggaatcgacgagacag ctcctgacaagagaaaggacgtggagaaggagatggctgagagagctaaggagagcatcgacagcctggagagcgctaaccctaagctgtacgctaact acatcaagtacagcgacgagaagaaggctgaggagttcacaagacagatcaacagagagaaggctaagacagctctgaacgcttacctgagaaacacaa agtggaacgtgatcatcagagaggacctgctgagaatcgacaacaagacatgtacactgttcagaaacaaggctgtgcacctggaggtggctagatacgtg cacgcttacatcaacgacatcgctgaggtgaacagctacttccagctgtaccactacatcatgcagagaatcatcatgaacgagagatacgagaagagcagc ggaaaggtgagcgagtacttcgacgctgtgaacgacgagaagaagtacaacgacagactgctgaagctgctgtgtgtgcctttcggatactgtatccctagat tcaagaacctgagcatcgaggctctgttcgacagaaacgaggctgctaagttcgacaaggagaagaagaaggtgagcggaaacagctga。

SEQ ID No.8：

atgatcgagaagaagaagagcttcgccaagggcatgggcgtgaagagcaccctggtgagcggcagcaaggtgtacatgaccaccttcgccgagg gcagcgatgcccgcctggagaagatcgtggagggcgatagcatccgcagcgtgaacgagggcgaggccttcagcgccgagatggccgataagaacgc cggctacaagatcggcaacgccaagttcagccaccccaagggctacgccgtggtggccaacaaccccctgtacaccggccccgtgcagcaggatatgct gggcctgaaggagaccctggagaagcgctacttcggcgagagcgccgatggcaacgataacatctgcatccaggtgatccacaacatcctggatatcgag aagatcctggccgagtacatcaccaacgccgcctacgccgtgaacaacatcagcggcctggataaggatatcatcggcttcggcaagttcagcaccgtgtac acctacgatgagttcaaggatcccgagcaccaccgcgccgccttcaacaacaacgataagctgatcaacgccatcaaggcccagtacgatgagttcgataa cttcctggataacccccgcctgggctacttcggccaggccttcttcagcaaggagggccgcaactacatcatcaactacggcaacgagtgctacgatatcctg gccctgctgagcggcctgcgccactgggtggtgcacaacaacgaggaggagagccgcatcagccgcacctggctgtacaacctggataagaacctggat aacgagtacatcagcaccctgaactacctgtacgatcgcatcaccaacgagctgaccaacagcttcagcaagaacagcgccgccaacgtgaactacatcgc cgagaccctgggcatcaaccccgccgagttcgccgagcagtacttccgcttcagcatcatgaaggagcagaagaacctgggcttcaacatcaccaagctgc gcgaggtgatgctggatcgcaaggatatgagcgagatccgcaagaaccacaaggtgttcgatagcatccgcaccaaggtgtacaccatgatggatttcgtga tctaccgctactacatcgaggaggatgccaaggtggccgccgccaacaagagcctgcccgataacgagaagagcctgagcgagaaggatatcttcgtgat caacctgcgcggcagcttcaacgatgatcagaaggatgccctgtactacgatgaggccaaccgcatctggcgcaagctggagaacatcatgcacaacatca aggagttccgcggcaacaagacccgcgagtacaagaagaaggatgccccccgcctgccccgcatcctgcccgccggccgcgatgtgagcgccttcagc aagctgatgtacgccctgaccatgttcctggatggcaaggagatcaacgatctgctgaccaccctgatcaacaagttcgataacatccagagcttcctgaaggt gatgcccctgatcggcgtgaacgccaagttcgtggaggagtacgccttcttcaaggatagcgccaagatcgccgatgagctgcgcctgatcaagagcttcgc ccgcatgggcgagcccatcgccgatgcccgccgcgccatgtacatcgatgccatccgcatcctgggcaccaacctgagctacgatgagctgaaggccctg gccgataccttcagcctggatgagaacggcaacaagctgaagaagggcaagcacggcatgcgcaacttcatcatcaacaacgtgatcagcaacaagcgct tccactacctgatccgctacggcgatcccgcccacctgcacgagatcgccaagaacgaggccgtggtgaagttcgtgctgggccgcatcgccgatatccag aagaagcagggccagaacggcaagaaccagatcgatcgctactacgagacctgcatcggcaaggataagggcaagagcgtgagcgagaaggtggatg ccctgaccaagatcatcaccggcatgaactacgatcagttcgataagaagcgcagcgtgatcgaggataccggccgcgagaacgccgagcgcgagaagtt caagaagatcatcagcctgtacctgaccgtgatctaccacatcctgaagaacatcgtgaacatcaacgcccgctacgtgatcggcttccactgcgtggagcgc gatgcccagctgtacaaggagaagggctacgatatcaacctgaagaagctggaggagaagggcttcagcagcgtgaccaagctgtgcgccggcatcgat gagaccgcccccgataagcgcaaggatgtggagaaggagatggccgagcgcgccaaggagagcatcgatagcctggagagcgccaaccccaagctgt acgccaactacatcaagtacagcgatgagaagaaggccgaggagttcacccgccagatcaaccgcgagaaggccaagaccgccctgaacgcctacctgc gcaacaccaagtggaacgtgatcatccgcgaggatctgctgcgcatcgataacaagacctgcaccctgttccgcaacaaggccgtgcacctggaggtggc ccgctacgtgcacgcctacatcaacgatatcgccgaggtgaacagctacttccagctgtaccactacatcatgcagcgcatcatcatgaacgagcgctacga gaagagcagcggcaaggtgagcgagtacttcgatgccgtgaacgatgagaagaagtacaacgatcgcctgctgaagctgctgtgcgtgcccttcggctact gcatcccccgcttcaagaacctgagcatcgaggccctgttcgatcgcaacgaggccgccaagttcgataaggagaagaagaaggtgagcggcaacagct ga。

SEQ ID No.9：

atgattgaaaaaaaaaaaagctttgcgaaaggcatgggcgtgaaaagcaccctggtgagcggcagcaaagtgtatatgaccacctttgcggaaggc agcgatgcgcgcctggaaaaaattgtggaaggcgatagcattcgcagcgtgaacgaaggcgaagcgtttagcgcggaaatggcggataaaaacgcgggc tataaaattggcaacgcgaaatttagccatccgaaaggctatgcggtggtggcgaacaacccgctgtataccggcccggtgcagcaggatatgctgggcctg aaagaaaccctggaaaaacgctattttggcgaaagcgcggatggcaacgataacatttgcattcaggtgattcataacattctggatattgaaaaaattctggcg gaatatattaccaacgcggcgtatgcggtgaacaacattagcggcctggataaagatattattggctttggcaaatttagcaccgtgtatacctatgatgaatttaa agatccggaacatcatcgcgcggcgtttaacaacaacgataaactgattaacgcgattaaagcgcagtatgatgaatttgataactttctggataacccgcgcct gggctattttggccaggcgttttttagcaaagaaggccgcaactatattattaactatggcaacgaatgctatgatattctggcgctgctgagcggcctgcgccat tgggtggtgcataacaacgaagaagaaagccgcattagccgcacctggctgtataacctggataaaaacctggataacgaatatattagcaccctgaactatc tgtatgatcgcattaccaacgaactgaccaacagctttagcaaaaacagcgcggcgaacgtgaactatattgcggaaaccctgggcattaacccggcggaat ttgcggaacagtattttcgctttagcattatgaaagaacagaaaaacctgggctttaacattaccaaactgcgcgaagtgatgctggatcgcaaagatatgagcg aaattcgcaaaaaccataaagtgtttgatagcattcgcaccaaagtgtataccatgatggattttgtgatttatcgctattatattgaagaagatgcgaaagtggcg gcggcgaacaaaagcctgccggataacgaaaaaagcctgagcgaaaaagatatttttgtgattaacctgcgcggcagctttaacgatgatcagaaagatgcg ctgtattatgatgaagcgaaccgcatttggcgcaaactggaaaacattatgcataacattaaagaatttcgcggcaacaaaacccgcgaatataaaaaaaaag atgcgccgcgcctgccgcgcattctgccggcgggccgcgatgtgagcgcgtttagcaaactgatgtatgcgctgaccatgtttctggatggcaaagaaattaa cgatctgctgaccaccctgattaacaaatttgataacattcagagctttctgaaagtgatgccgctgattggcgtgaacgcgaaatttgtggaagaatatgcgtttt ttaaagatagcgcgaaaattgcggatgaactgcgcctgattaaaagctttgcgcgcatgggcgaaccgattgcggatgcgcgccgcgcgatgtatattgatgc gattcgcattctgggcaccaacctgagctatgatgaactgaaagcgctggcggatacctttagcctggatgaaaacggcaacaaactgaaaaaaggcaaaca tggcatgcgcaactttattattaacaacgtgattagcaacaaacgctttcattatctgattcgctatggcgatccggcgcatctgcatgaaattgcgaaaaacgaa gcggtggtgaaatttgtgctgggccgcattgcggatattcagaaaaaacagggccagaacggcaaaaaccagattgatcgctattatgaaacctgcattggca aagataaaggcaaaagcgtgagcgaaaaagtggatgcgctgaccaaaattattaccggcatgaactatgatcagtttgataaaaaacgcagcgtgattgaag ataccggccgcgaaaacgcggaacgcgaaaaatttaaaaaaattattagcctgtatctgaccgtgatttatcatattctgaaaaacattgtgaacattaacgcgc gctatgtgattggctttcattgcgtggaacgcgatgcgcagctgtataaagaaaaaggctatgatattaacctgaaaaaactggaagaaaaaggctttagcagc gtgaccaaactgtgcgcgggcattgatgaaaccgcgccggataaacgcaaagatgtggaaaaagaaatggcggaacgcgcgaaagaaagcattgatagc ctggaaagcgcgaacccgaaactgtatgcgaactatattaaatatagcgatgaaaaaaaagcggaagaatttacccgccagattaaccgcgaaaaagcgaa aaccgcgctgaacgcgtatctgcgcaacaccaaatggaacgtgattattcgcgaagatctgctgcgcattgataacaaaacctgcaccctgtttcgcaacaaa gcggtgcatctggaagtggcgcgctatgtgcatgcgtatattaacgatattgcggaagtgaacagctattttcagctgtatcattatattatgcagcgcattattatg aacgaacgctatgaaaaaagcagcggcaaagtgagcgaatattttgatgcggtgaacgatgaaaaaaaatataacgatcgcctgctgaaactgctgtgcgtg ccgtttggctattgcattccgcgctttaaaaacctgagcattgaagcgctgtttgatcgcaacgaagcggcgaaatttgataaagaaaaaaaaaaagtgagcgg caacagctga。

SEQ ID No.10：

atgatcgagaagaagaagagcttcgccaagggcatgggcgtgaagagcaccctggtgagcggcagcaaggtgtacatgaccaccttcgccgagg gcagcgacgccaggctggagaagatcgtggagggcgacagcatcaggagcgtgaacgagggcgaggccttcagcgccgagatggccgacaagaacg ccggctacaagatcggcaacgccaagttcagccaccccaagggctacgccgtggtggccaacaaccccctgtacaccggccccgtgcagcaggacatgc tgggcctgaaggagaccctggagaagaggtacttcggcgagagcgccgacggcaacgacaacatctgcatccaggtgatccacaacatcctggacatcg agaagatcctggccgagtacatcaccaacgccgcctacgccgtgaacaacatcagcggcctggacaaggacatcatcggcttcggcaagttcagcaccgt gtacacctacgacgagttcaaggaccccgagcaccacagggccgccttcaacaacaacgacaagctgatcaacgccatcaaggcccagtacgacgagttc gacaacttcctggacaaccccaggctgggctacttcggccaggccttcttcagcaaggagggcaggaactacatcatcaactacggcaacgagtgctacga catcctggccctgctgagcggcctgaggcactgggtggtgcacaacaacgaggaggagagcaggatcagcaggacctggctgtacaacctggacaagaa cctggacaacgagtacatcagcaccctgaactacctgtacgacaggatcaccaacgagctgaccaacagcttcagcaagaacagcgccgccaacgtgaac tacatcgccgagaccctgggcatcaaccccgccgagttcgccgagcagtacttcaggttcagcatcatgaaggagcagaagaacctgggcttcaacatcac caagctgagggaggtgatgctggacaggaaggacatgagcgagatcaggaagaaccacaaggtgttcgacagcatcaggaccaaggtgtacaccatgat ggacttcgtgatctacaggtactacatcgaggaggacgccaaggtggccgccgccaacaagagcctgcccgacaacgagaagagcctgagcgagaagg acatcttcgtgatcaacctgaggggcagcttcaacgacgaccagaaggacgccctgtactacgacgaggccaacaggatctggaggaagctggagaacat catgcacaacatcaaggagttcaggggcaacaagaccagggagtacaagaagaaggacgcccccaggctgcccaggatcctgcccgccggcagggac gtgagcgccttcagcaagctgatgtacgccctgaccatgttcctggacggcaaggagatcaacgacctgctgaccaccctgatcaacaagttcgacaacatc cagagcttcctgaaggtgatgcccctgatcggcgtgaacgccaagttcgtggaggagtacgccttcttcaaggacagcgccaagatcgccgacgagctgag gctgatcaagagcttcgccaggatgggcgagcccatcgccgacgccaggagggccatgtacatcgacgccatcaggatcctgggcaccaacctgagcta cgacgagctgaaggccctggccgacaccttcagcctggacgagaacggcaacaagctgaagaagggcaagcacggcatgaggaacttcatcatcaacaa cgtgatcagcaacaagaggttccactacctgatcaggtacggcgaccccgcccacctgcacgagatcgccaagaacgaggccgtggtgaagttcgtgctg ggcaggatcgccgacatccagaagaagcagggccagaacggcaagaaccagatcgacaggtactacgagacctgcatcggcaaggacaagggcaaga gcgtgagcgagaaggtggacgccctgaccaagatcatcaccggcatgaactacgaccagttcgacaagaagaggagcgtgatcgaggacaccggcagg gagaacgccgagagggagaagttcaagaagatcatcagcctgtacctgaccgtgatctaccacatcctgaagaacatcgtgaacatcaacgccaggtacgt gatcggcttccactgcgtggagagggacgcccagctgtacaaggagaagggctacgacatcaacctgaagaagctggaggagaagggcttcagcagcgt gaccaagctgtgcgccggcatcgacgagaccgcccccgacaagaggaaggacgtggagaaggagatggccgagagggccaaggagagcatcgacag cctggagagcgccaaccccaagctgtacgccaactacatcaagtacagcgacgagaagaaggccgaggagttcaccaggcagatcaacagggagaagg ccaagaccgccctgaacgcctacctgaggaacaccaagtggaacgtgatcatcagggaggacctgctgaggatcgacaacaagacctgcaccctgttcag gaacaaggccgtgcacctggaggtggccaggtacgtgcacgcctacatcaacgacatcgccgaggtgaacagctacttccagctgtaccactacatcatgc agaggatcatcatgaacgagaggtacgagaagagcagcggcaaggtgagcgagtacttcgacgccgtgaacgacgagaagaagtacaacgacaggctg ctgaagctgctgtgcgtgcccttcggctactgcatccccaggttcaagaacctgagcatcgaggccctgttcgacaggaacgaggccgccaagttcgacaa ggagaagaagaaggtgagcggcaacagctga。

SEQ ID No.11：

atgatcgagaagaagaagagcttcgccaagggcatgggcgtgaagagcaccctggtgagcggcagcaaggtgtacatgaccaccttcgccgagg gcagcgacgccaggctggagaagatcgtggagggcgacagcatcaggagcgtgaacgagggcgaggccttcagcgccgagatggccgacaagaacg ccggctacaagatcggcaacgccaagttcagccaccctaagggctacgccgtggtggccaacaaccctctgtacaccggccctgtgcagcaggacatgct gggcctgaaggagaccctggagaagaggtacttcggcgagagcgccgacggcaacgacaacatctgcatccaggtgatccacaacatcctggacatcga gaagatcctggccgagtacatcaccaacgccgcctacgccgtgaacaacatcagcggcctggacaaggacatcatcggcttcggcaagttcagcaccgtgt acacctacgacgagttcaaggaccctgagcaccacagggccgccttcaacaacaacgacaagctgatcaacgccatcaaggcccagtacgacgagttcga caacttcctggacaaccctaggctgggctacttcggccaggccttcttcagcaaggagggcaggaactacatcatcaactacggcaacgagtgctacgacat cctggccctgctgagcggcctgaggcactgggtggtgcacaacaacgaggaggagagcaggatcagcaggacctggctgtacaacctggacaagaacct ggacaacgagtacatcagcaccctgaactacctgtacgacaggatcaccaacgagctgaccaacagcttcagcaagaacagcgccgccaacgtgaactac atcgccgagaccctgggcatcaaccctgccgagttcgccgagcagtacttcaggttcagcatcatgaaggagcagaagaacctgggcttcaacatcaccaa gctgagggaggtgatgctggacaggaaggacatgagcgagatcaggaagaaccacaaggtgttcgacagcatcaggaccaaggtgtacaccatgatgga cttcgtgatctacaggtactacatcgaggaggacgccaaggtggccgccgccaacaagagcctgcctgacaacgagaagagcctgagcgagaaggacat cttcgtgatcaacctgaggggcagcttcaacgacgaccagaaggacgccctgtactacgacgaggccaacaggatctggaggaagctggagaacatcatg cacaacatcaaggagttcaggggcaacaagaccagggagtacaagaagaaggacgcccctaggctgcctaggatcctgcctgccggcagggacgtgag cgccttcagcaagctgatgtacgccctgaccatgttcctggacggcaaggagatcaacgacctgctgaccaccctgatcaacaagttcgacaacatccagag cttcctgaaggtgatgcctctgatcggcgtgaacgccaagttcgtggaggagtacgccttcttcaaggacagcgccaagatcgccgacgagctgaggctgat caagagcttcgccaggatgggcgagcctatcgccgacgccaggagggccatgtacatcgacgccatcaggatcctgggcaccaacctgagctacgacga gctgaaggccctggccgacaccttcagcctggacgagaacggcaacaagctgaagaagggcaagcacggcatgaggaacttcatcatcaacaacgtgat cagcaacaagaggttccactacctgatcaggtacggcgaccctgcccacctgcacgagatcgccaagaacgaggccgtggtgaagttcgtgctgggcagg atcgccgacatccagaagaagcagggccagaacggcaagaaccagatcgacaggtactacgagacctgcatcggcaaggacaagggcaagagcgtga gcgagaaggtggacgccctgaccaagatcatcaccggcatgaactacgaccagttcgacaagaagaggagcgtgatcgaggacaccggcagggagaac gccgagagggagaagttcaagaagatcatcagcctgtacctgaccgtgatctaccacatcctgaagaacatcgtgaacatcaacgccaggtacgtgatcgg cttccactgcgtggagagggacgcccagctgtacaaggagaagggctacgacatcaacctgaagaagctggaggagaagggcttcagcagcgtgaccaa gctgtgcgccggcatcgacgagaccgcccctgacaagaggaaggacgtggagaaggagatggccgagagggccaaggagagcatcgacagcctgga gagcgccaaccctaagctgtacgccaactacatcaagtacagcgacgagaagaaggccgaggagttcaccaggcagatcaacagggagaaggccaaga ccgccctgaacgcctacctgaggaacaccaagtggaacgtgatcatcagggaggacctgctgaggatcgacaacaagacctgcaccctgttcaggaacaa ggccgtgcacctggaggtggccaggtacgtgcacgcctacatcaacgacatcgccgaggtgaacagctacttccagctgtaccactacatcatgcagagga tcatcatgaacgagaggtacgagaagagcagcggcaaggtgagcgagtacttcgacgccgtgaacgacgagaagaagtacaacgacaggctgctgaag ctgctgtgcgtgcctttcggctactgcatccctaggttcaagaacctgagcatcgaggccctgttcgacaggaacgaggccgccaagttcgacaaggagaag aagaaggtgagcggcaacagctga。

SEQ ID No.12：

atgattgaaaagaagaagtcttttgctaagggaatgggagttaagtctactcttgtttctggatctaaggtttatatgactacttttgctgaaggatctgatgc tagacttgaaaagattgttgaaggagattctattagatctgttaatgaaggagaagctttttctgctgaaatggctgataagaatgctggatataagattggaaatg ctaagttttctcatccaaagggatatgctgttgttgctaataatccactttatactggaccagttcaacaagatatgcttggacttaaggaaactcttgaaaagagat attttggagaatctgctgatggaaatgataatatttgtattcaagttattcataatattcttgatattgaaaagattcttgctgaatatattactaatgctgcttatgctgtta ataatatttctggacttgataaggatattattggatttggaaagttttctactgtttatacttatgatgaatttaaggatccagaacatcatagagctgcttttaataataa tgataagcttattaatgctattaaggctcaatatgatgaatttgataattttcttgataatccaagacttggatattttggacaagcttttttttctaaggaaggaagaaat tatattattaattatggaaatgaatgttatgatattcttgctcttctttctggacttagacattgggttgttcataataatgaagaagaatctagaatttctagaacttggct ttataatcttgataagaatcttgataatgaatatatttctactcttaattatctttatgatagaattactaatgaacttactaattctttttctaagaattctgctgctaatgtta attatattgctgaaactcttggaattaatccagctgaatttgctgaacaatattttagattttctattatgaaggaacaaaagaatcttggatttaatattactaagcttag agaagttatgcttgatagaaaggatatgtctgaaattagaaagaatcataaggtttttgattctattagaactaaggtttatactatgatggattttgttatttatagatat tatattgaagaagatgctaaggttgctgctgctaataagtctcttccagataatgaaaagtctctttctgaaaaggatatttttgttattaatcttagaggatcttttaat gatgatcaaaaggatgctctttattatgatgaagctaatagaatttggagaaagcttgaaaatattatgcataatattaaggaatttagaggaaataagactagaga atataagaagaaggatgctccaagacttccaagaattcttccagctggaagagatgtttctgctttttctaagcttatgtatgctcttactatgtttcttgatggaaag gaaattaatgatcttcttactactcttattaataagtttgataatattcaatcttttcttaaggttatgccacttattggagttaatgctaagtttgttgaagaatatgctttttt taaggattctgctaagattgctgatgaacttagacttattaagtcttttgctagaatgggagaaccaattgctgatgctagaagagctatgtatattgatgctattaga attcttggaactaatctttcttatgatgaacttaaggctcttgctgatactttttctcttgatgaaaatggaaataagcttaagaagggaaagcatggaatgagaaatt ttattattaataatgttatttctaataagagatttcattatcttattagatatggagatccagctcatcttcatgaaattgctaagaatgaagctgttgttaagtttgttcttg gaagaattgctgatattcaaaagaagcaaggacaaaatggaaagaatcaaattgatagatattatgaaacttgtattggaaaggataagggaaagtctgtttctg aaaaggttgatgctcttactaagattattactggaatgaattatgatcaatttgataagaagagatctgttattgaagatactggaagagaaaatgctgaaagagaa aagtttaagaagattatttctctttatcttactgttatttatcatattcttaagaatattgttaatattaatgctagatatgttattggatttcattgtgttgaaagagatgctca actttataaggaaaagggatatgatattaatcttaagaagcttgaagaaaagggattttcttctgttactaagctttgtgctggaattgatgaaactgctccagataa gagaaaggatgttgaaaaggaaatggctgaaagagctaaggaatctattgattctcttgaatctgctaatccaaagctttatgctaattatattaagtattctgatga aaagaaggctgaagaatttactagacaaattaatagagaaaaggctaagactgctcttaatgcttatcttagaaatactaagtggaatgttattattagagaagatc ttcttagaattgataataagacttgtactctttttagaaataaggctgttcatcttgaagttgctagatatgttcatgcttatattaatgatattgctgaagttaattcttattt tcaactttatcattatattatgcaaagaattattatgaatgaaagatatgaaaagtcttctggaaaggtttctgaatattttgatgctgttaatgatgaaaagaagtataa tgatagacttcttaagcttctttgtgttccatttggatattgtattccaagatttaagaatctttctattgaagctctttttgatagaaatgaagctgctaagtttgataagg aaaagaagaaggtttctggaaattcttga。

SEQ ID No.13：

atgattgaaaagaagaagtcttttgctaagggtatgggtgttaagtctactttggtttctggttctaaggtttacatgactacttttgctgaaggttctgatgct agattggaaaagattgttgaaggtgattctattagatctgttaacgaaggtgaagctttttctgctgaaatggctgataagaacgctggttacaagattggtaacgc taagttttctcatccaaagggttacgctgttgttgctaacaacccattgtacactggtccagttcaacaagatatgttgggtttgaaggaaactttggaaaagagat actttggtgaatctgctgatggtaacgataacatttgtattcaagttattcataacattttggatattgaaaagattttggctgaatacattactaacgctgcttacgctg ttaacaacatttctggtttggataaggatattattggttttggtaagttttctactgtttacacttacgatgaatttaaggatccagaacatcatagagctgcttttaacaa caacgataagttgattaacgctattaaggctcaatacgatgaatttgataactttttggataacccaagattgggttactttggtcaagcttttttttctaaggaaggta gaaactacattattaactacggtaacgaatgttacgatattttggctttgttgtctggtttgagacattgggttgttcataacaacgaagaagaatctagaatttctag aacttggttgtacaacttggataagaacttggataacgaatacatttctactttgaactacttgtacgatagaattactaacgaattgactaactctttttctaagaact ctgctgctaacgttaactacattgctgaaactttgggtattaacccagctgaatttgctgaacaatactttagattttctattatgaaggaacaaaagaacttgggtttt aacattactaagttgagagaagttatgttggatagaaaggatatgtctgaaattagaaagaaccataaggtttttgattctattagaactaaggtttacactatgatg gattttgttatttacagatactacattgaagaagatgctaaggttgctgctgctaacaagtctttgccagataacgaaaagtctttgtctgaaaaggatatttttgttatt aacttgagaggttcttttaacgatgatcaaaaggatgctttgtactacgatgaagctaacagaatttggagaaagttggaaaacattatgcataacattaaggaatt tagaggtaacaagactagagaatacaagaagaaggatgctccaagattgccaagaattttgccagctggtagagatgtttctgctttttctaagttgatgtacgct ttgactatgtttttggatggtaaggaaattaacgatttgttgactactttgattaacaagtttgataacattcaatcttttttgaaggttatgccattgattggtgttaacgc taagtttgttgaagaatacgctttttttaaggattctgctaagattgctgatgaattgagattgattaagtcttttgctagaatgggtgaaccaattgctgatgctagaa gagctatgtacattgatgctattagaattttgggtactaacttgtcttacgatgaattgaaggctttggctgatactttttctttggatgaaaacggtaacaagttgaag aagggtaagcatggtatgagaaactttattattaacaacgttatttctaacaagagatttcattacttgattagatacggtgatccagctcatttgcatgaaattgcta agaacgaagctgttgttaagtttgttttgggtagaattgctgatattcaaaagaagcaaggtcaaaacggtaagaaccaaattgatagatactacgaaacttgtatt ggtaaggataagggtaagtctgtttctgaaaaggttgatgctttgactaagattattactggtatgaactacgatcaatttgataagaagagatctgttattgaagat actggtagagaaaacgctgaaagagaaaagtttaagaagattatttctttgtacttgactgttatttaccatattttgaagaacattgttaacattaacgctagatacg ttattggttttcattgtgttgaaagagatgctcaattgtacaaggaaaagggttacgatattaacttgaagaagttggaagaaaagggtttttcttctgttactaagttg tgtgctggtattgatgaaactgctccagataagagaaaggatgttgaaaaggaaatggctgaaagagctaaggaatctattgattctttggaatctgctaaccca aagttgtacgctaactacattaagtactctgatgaaaagaaggctgaagaatttactagacaaattaacagagaaaaggctaagactgctttgaacgcttacttg agaaacactaagtggaacgttattattagagaagatttgttgagaattgataacaagacttgtactttgtttagaaacaaggctgttcatttggaagttgctagatac gttcatgcttacattaacgatattgctgaagttaactcttactttcaattgtaccattacattatgcaaagaattattatgaacgaaagatacgaaaagtcttctggtaa ggtttctgaatactttgatgctgttaacgatgaaaagaagtacaacgatagattgttgaagttgttgtgtgttccatttggttactgtattccaagatttaagaacttgtc tattgaagctttgtttgatagaaacgaagctgctaagtttgataaggaaaagaagaaggtttctggtaactcttga。

SEQ ID No.14：

SEQ ID No.15：

atgattgaaaaaaaaaaatcttttgctaaaggtatgggtgttaaatctactttggtttctggttctaaagtttatatgactacttttgctgaaggttctgatgcta gattggaaaaaattgttgaaggtgattctattagatctgttaatgaaggtgaagctttttctgctgaaatggctgataaaaatgctggttataaaattggtaatgctaa attttctcatccaaaaggttatgctgttgttgctaataatccattgtatactggtccagttcaacaagatatgttgggtttgaaagaaactttggaaaaaagatattttg gtgaatctgctgatggtaatgataatatttgtattcaagttattcataatattttggatattgaaaaaattttggctgaatatattactaatgctgcttatgctgttaataata tttctggtttggataaagatattattggttttggtaaattttctactgtttatacttatgatgaatttaaagatccagaacatcatagagctgcttttaataataatgataaat tgattaatgctattaaagctcaatatgatgaatttgataattttttggataatccaagattgggttattttggtcaagcttttttttctaaagaaggtagaaattatattatta attatggtaatgaatgttatgatattttggctttgttgtctggtttgagacattgggttgttcataataatgaagaagaatctagaatttctagaacttggttgtataatttg gataaaaatttggataatgaatatatttctactttgaattatttgtatgatagaattactaatgaattgactaattctttttctaaaaattctgctgctaatgttaattatattg ctgaaactttgggtattaatccagctgaatttgctgaacaatattttagattttctattatgaaagaacaaaaaaatttgggttttaatattactaaattgagagaagtta tgttggatagaaaagatatgtctgaaattagaaaaaatcataaagtttttgattctattagaactaaagtttatactatgatggattttgttatttatagatattatattgaa gaagatgctaaagttgctgctgctaataaatctttgccagataatgaaaaatctttgtctgaaaaagatatttttgttattaatttgagaggttcttttaatgatgatcaa aaagatgctttgtattatgatgaagctaatagaatttggagaaaattggaaaatattatgcataatattaaagaatttagaggtaataaaactagagaatataaaaaa aaagatgctccaagattgccaagaattttgccagctggtagagatgtttctgctttttctaaattgatgtatgctttgactatgtttttggatggtaaagaaattaatgat ttgttgactactttgattaataaatttgataatattcaatcttttttgaaagttatgccattgattggtgttaatgctaaatttgttgaagaatatgctttttttaaagattctgc taaaattgctgatgaattgagattgattaaatcttttgctagaatgggtgaaccaattgctgatgctagaagagctatgtatattgatgctattagaattttgggtacta atttgtcttatgatgaattgaaagctttggctgatactttttctttggatgaaaatggtaataaattgaaaaaaggtaaacatggtatgagaaattttattattaataatgt tatttctaataaaagatttcattatttgattagatatggtgatccagctcatttgcatgaaattgctaaaaatgaagctgttgttaaatttgttttgggtagaattgctgata ttcaaaaaaaacaaggtcaaaatggtaaaaatcaaattgatagatattatgaaacttgtattggtaaagataaaggtaaatctgtttctgaaaaagttgatgctttga ctaaaattattactggtatgaattatgatcaatttgataaaaaaagatctgttattgaagatactggtagagaaaatgctgaaagagaaaaatttaaaaaaattatttc tttgtatttgactgttatttatcatattttgaaaaatattgttaatattaatgctagatatgttattggttttcattgtgttgaaagagatgctcaattgtataaagaaaaaggt tatgatattaatttgaaaaaattggaagaaaaaggtttttcttctgttactaaattgtgtgctggtattgatgaaactgctccagataaaagaaaagatgttgaaaaag aaatggctgaaagagctaaagaatctattgattctttggaatctgctaatccaaaattgtatgctaattatattaaatattctgatgaaaaaaaagctgaagaatttac tagacaaattaatagagaaaaagctaaaactgctttgaatgcttatttgagaaatactaaatggaatgttattattagagaagatttgttgagaattgataataaaact tgtactttgtttagaaataaagctgttcatttggaagttgctagatatgttcatgcttatattaatgatattgctgaagttaattcttattttcaattgtatcattatattatgca aagaattattatgaatgaaagatatgaaaaatcttctggtaaagtttctgaatattttgatgctgttaatgatgaaaaaaaatataatgatagattgttgaaattgttgtg tgttccatttggttattgtattccaagatttaaaaatttgtctattgaagctttgtttgatagaaatgaagctgctaaatttgataaagaaaaaaaaaaagtttctggtaat tcttga。

SEQ ID No.16：

atgattgaaaaaaaaaaatcttttgctaaaggtatgggtgttaaatctactttagtttctggttctaaagtttatatgactacttttgctgaaggttctgatgctcg tttagaaaaaattgttgaaggtgattctattcgttctgttaatgaaggtgaagctttttctgctgaaatggctgataaaaatgctggttataaaattggtaatgctaaatt ttctcatcctaaaggttatgctgttgttgctaataatcctttatatactggtcctgttcaacaagatatgttaggtttaaaagaaactttagaaaaacgttattttggtgaa tctgctgatggtaatgataatatttgtattcaagttattcataatattttagatattgaaaaaattttagctgaatatattactaatgctgcttatgctgttaataatatttctg gtttagataaagatattattggttttggtaaattttctactgtttatacttatgatgaatttaaagatcctgaacatcatcgtgctgcttttaataataatgataaattaatta atgctattaaagctcaatatgatgaatttgataattttttagataatcctcgtttaggttattttggtcaagcttttttttctaaagaaggtcgtaattatattattaattatggt aatgaatgttatgatattttagctttattatctggtttacgtcattgggttgttcataataatgaagaagaatctcgtatttctcgtacttggttatataatttagataaaaat ttagataatgaatatatttctactttaaattatttatatgatcgtattactaatgaattaactaattctttttctaaaaattctgctgctaatgttaattatattgctgaaacttta ggtattaatcctgctgaatttgctgaacaatattttcgtttttctattatgaaagaacaaaaaaatttaggttttaatattactaaattacgtgaagttatgttagatcgtaa agatatgtctgaaattcgtaaaaatcataaagtttttgattctattcgtactaaagtttatactatgatggattttgttatttatcgttattatattgaagaagatgctaaagt tgctgctgctaataaatctttacctgataatgaaaaatctttatctgaaaaagatatttttgttattaatttacgtggttcttttaatgatgatcaaaaagatgctttatatta tgatgaagctaatcgtatttggcgtaaattagaaaatattatgcataatattaaagaatttcgtggtaataaaactcgtgaatataaaaaaaaagatgctcctcgttta cctcgtattttacctgctggtcgtgatgtttctgctttttctaaattaatgtatgctttaactatgtttttagatggtaaagaaattaatgatttattaactactttaattaataa atttgataatattcaatcttttttaaaagttatgcctttaattggtgttaatgctaaatttgttgaagaatatgctttttttaaagattctgctaaaattgctgatgaattacgtt taattaaatcttttgctcgtatgggtgaacctattgctgatgctcgtcgtgctatgtatattgatgctattcgtattttaggtactaatttatcttatgatgaattaaaagctt tagctgatactttttctttagatgaaaatggtaataaattaaaaaaaggtaaacatggtatgcgtaattttattattaataatgttatttctaataaacgttttcattatttaat tcgttatggtgatcctgctcatttacatgaaattgctaaaaatgaagctgttgttaaatttgttttaggtcgtattgctgatattcaaaaaaaacaaggtcaaaatggta aaaatcaaattgatcgttattatgaaacttgtattggtaaagataaaggtaaatctgtttctgaaaaagttgatgctttaactaaaattattactggtatgaattatgatc aatttgataaaaaacgttctgttattgaagatactggtcgtgaaaatgctgaacgtgaaaaatttaaaaaaattatttctttatatttaactgttatttatcatattttaaaa aatattgttaatattaatgctcgttatgttattggttttcattgtgttgaacgtgatgctcaattatataaagaaaaaggttatgatattaatttaaaaaaattagaagaaa aaggtttttcttctgttactaaattatgtgctggtattgatgaaactgctcctgataaacgtaaagatgttgaaaaagaaatggctgaacgtgctaaagaatctattga ttctttagaatctgctaatcctaaattatatgctaattatattaaatattctgatgaaaaaaaagctgaagaatttactcgtcaaattaatcgtgaaaaagctaaaactg ctttaaatgcttatttacgtaatactaaatggaatgttattattcgtgaagatttattacgtattgataataaaacttgtactttatttcgtaataaagctgttcatttagaag ttgctcgttatgttcatgcttatattaatgatattgctgaagttaattcttattttcaattatatcattatattatgcaacgtattattatgaatgaacgttatgaaaaatcttct ggtaaagtttctgaatattttgatgctgttaatgatgaaaaaaaatataatgatcgtttattaaaattattatgtgttccttttggttattgtattcctcgttttaaaaatttatc tattgaagctttatttgatcgtaatgaagctgctaaatttgataaagaaaaaaaaaaagtttctggtaattcttga。

SEQ ID No.17：

atgatcgagaagaagaagtccttcgctaagggtatgggtgtgaagtccaccctggtgtccggttccaaggtgtacatgaccaccttcgctgagggttcc gacgctcgtctggagaagatcgtggagggtgactccatccgttccgtgaacgagggtgaggctttctccgctgagatggctgacaagaacgctggttacaag atcggtaacgctaagttctcccaccctaagggttacgctgtggtggctaacaaccctctgtacaccggtcctgtgcagcaggacatgctgggtctgaaggaga ccctggagaagcgttacttcggtgagtccgctgacggtaacgacaacatctgcatccaggtgatccacaacatcctggacatcgagaagatcctggctgagt acatcaccaacgctgcttacgctgtgaacaacatctccggtctggacaaggacatcatcggtttcggtaagttctccaccgtgtacacctacgacgagttcaag gaccctgagcaccaccgtgctgctttcaacaacaacgacaagctgatcaacgctatcaaggctcagtacgacgagttcgacaacttcctggacaaccctcgtc tgggttacttcggtcaggctttcttctccaaggagggtcgtaactacatcatcaactacggtaacgagtgctacgacatcctggctctgctgtccggtctgcgtca ctgggtggtgcacaacaacgaggaggagtcccgtatctcccgtacctggctgtacaacctggacaagaacctggacaacgagtacatctccaccctgaacta cctgtacgaccgtatcaccaacgagctgaccaactccttctccaagaactccgctgctaacgtgaactacatcgctgagaccctgggtatcaaccctgctgagt tcgctgagcagtacttccgtttctccatcatgaaggagcagaagaacctgggtttcaacatcaccaagctgcgtgaggtgatgctggaccgtaaggacatgtc cgagatccgtaagaaccacaaggtgttcgactccatccgtaccaaggtgtacaccatgatggacttcgtgatctaccgttactacatcgaggaggacgctaag gtggctgctgctaacaagtccctgcctgacaacgagaagtccctgtccgagaaggacatcttcgtgatcaacctgcgtggttccttcaacgacgaccagaag gacgctctgtactacgacgaggctaaccgtatctggcgtaagctggagaacatcatgcacaacatcaaggagttccgtggtaacaagacccgtgagtacaag aagaaggacgctcctcgtctgcctcgtatcctgcctgctggtcgtgacgtgtccgctttctccaagctgatgtacgctctgaccatgttcctggacggtaaggag atcaacgacctgctgaccaccctgatcaacaagttcgacaacatccagtccttcctgaaggtgatgcctctgatcggtgtgaacgctaagttcgtggaggagta cgctttcttcaaggactccgctaagatcgctgacgagctgcgtctgatcaagtccttcgctcgtatgggtgagcctatcgctgacgctcgtcgtgctatgtacatc gacgctatccgtatcctgggtaccaacctgtcctacgacgagctgaaggctctggctgacaccttctccctggacgagaacggtaacaagctgaagaagggt aagcacggtatgcgtaacttcatcatcaacaacgtgatctccaacaagcgtttccactacctgatccgttacggtgaccctgctcacctgcacgagatcgctaa gaacgaggctgtggtgaagttcgtgctgggtcgtatcgctgacatccagaagaagcagggtcagaacggtaagaaccagatcgaccgttactacgagacct gcatcggtaaggacaagggtaagtccgtgtccgagaaggtggacgctctgaccaagatcatcaccggtatgaactacgaccagttcgacaagaagcgttcc gtgatcgaggacaccggtcgtgagaacgctgagcgtgagaagttcaagaagatcatctccctgtacctgaccgtgatctaccacatcctgaagaacatcgtg aacatcaacgctcgttacgtgatcggtttccactgcgtggagcgtgacgctcagctgtacaaggagaagggttacgacatcaacctgaagaagctggaggag aagggtttctcctccgtgaccaagctgtgcgctggtatcgacgagaccgctcctgacaagcgtaaggacgtggagaaggagatggctgagcgtgctaagga gtccatcgactccctggagtccgctaaccctaagctgtacgctaactacatcaagtactccgacgagaagaaggctgaggagttcacccgtcagatcaaccgt gagaaggctaagaccgctctgaacgcttacctgcgtaacaccaagtggaacgtgatcatccgtgaggacctgctgcgtatcgacaacaagacctgcaccct gttccgtaacaaggctgtgcacctggaggtggctcgttacgtgcacgcttacatcaacgacatcgctgaggtgaactcctacttccagctgtaccactacatcat gcagcgtatcatcatgaacgagcgttacgagaagtcctccggtaaggtgtccgagtacttcgacgctgtgaacgacgagaagaagtacaacgaccgtctgct gaagctgctgtgcgtgcctttcggttactgcatccctcgtttcaagaacctgtccatcgaggctctgttcgaccgtaacgaggctgctaagttcgacaaggagaa gaagaaggtgtccggtaactcctga。

SEQ ID No.18：

atgatcgagaagaagaagtccttcgccaagggcatgggcgtgaagtccaccctggtgtccggctccaaggtgtacatgaccaccttcgccgagggc tccgacgccaggctggagaagatcgtggagggcgactccatcaggtccgtgaacgagggcgaggccttctccgccgagatggccgacaagaacgccgg ctacaagatcggcaacgccaagttctcccacccgaagggctacgccgtggtggccaacaacccgctgtacaccggcccggtgcagcaggacatgctggg cctgaaggagaccctggagaagaggtacttcggcgagtccgccgacggcaacgacaacatctgcatccaggtgatccacaacatcctggacatcgagaag atcctggccgagtacatcaccaacgccgcctacgccgtgaacaacatctccggcctggacaaggacatcatcggcttcggcaagttctccaccgtgtacacc tacgacgagttcaaggacccggagcaccacagggccgccttcaacaacaacgacaagctgatcaacgccatcaaggcccagtacgacgagttcgacaact tcctggacaacccgaggctgggctacttcggccaggccttcttctccaaggagggcaggaactacatcatcaactacggcaacgagtgctacgacatcctgg ccctgctgtccggcctgaggcactgggtggtgcacaacaacgaggaggagtccaggatctccaggacctggctgtacaacctggacaagaacctggacaa cgagtacatctccaccctgaactacctgtacgacaggatcaccaacgagctgaccaactccttctccaagaactccgccgccaacgtgaactacatcgccga gaccctgggcatcaacccggccgagttcgccgagcagtacttcaggttctccatcatgaaggagcagaagaacctgggcttcaacatcaccaagctgaggg aggtgatgctggacaggaaggacatgtccgagatcaggaagaaccacaaggtgttcgactccatcaggaccaaggtgtacaccatgatggacttcgtgatct acaggtactacatcgaggaggacgccaaggtggccgccgccaacaagtccctgccggacaacgagaagtccctgtccgagaaggacatcttcgtgatcaa cctgaggggctccttcaacgacgaccagaaggacgccctgtactacgacgaggccaacaggatctggaggaagctggagaacatcatgcacaacatcaa ggagttcaggggcaacaagaccagggagtacaagaagaaggacgccccgaggctgccgaggatcctgccggccggcagggacgtgtccgccttctcca agctgatgtacgccctgaccatgttcctggacggcaaggagatcaacgacctgctgaccaccctgatcaacaagttcgacaacatccagtccttcctgaaggt gatgccgctgatcggcgtgaacgccaagttcgtggaggagtacgccttcttcaaggactccgccaagatcgccgacgagctgaggctgatcaagtccttcgc caggatgggcgagccgatcgccgacgccaggagggccatgtacatcgacgccatcaggatcctgggcaccaacctgtcctacgacgagctgaaggccct ggccgacaccttctccctggacgagaacggcaacaagctgaagaagggcaagcacggcatgaggaacttcatcatcaacaacgtgatctccaacaagagg ttccactacctgatcaggtacggcgacccggcccacctgcacgagatcgccaagaacgaggccgtggtgaagttcgtgctgggcaggatcgccgacatcc agaagaagcagggccagaacggcaagaaccagatcgacaggtactacgagacctgcatcggcaaggacaagggcaagtccgtgtccgagaaggtgga cgccctgaccaagatcatcaccggcatgaactacgaccagttcgacaagaagaggtccgtgatcgaggacaccggcagggagaacgccgagagggaga agttcaagaagatcatctccctgtacctgaccgtgatctaccacatcctgaagaacatcgtgaacatcaacgccaggtacgtgatcggcttccactgcgtggag agggacgcccagctgtacaaggagaagggctacgacatcaacctgaagaagctggaggagaagggcttctcctccgtgaccaagctgtgcgccggcatc gacgagaccgccccggacaagaggaaggacgtggagaaggagatggccgagagggccaaggagtccatcgactccctggagtccgccaacccgaag ctgtacgccaactacatcaagtactccgacgagaagaaggccgaggagttcaccaggcagatcaacagggagaaggccaagaccgccctgaacgcctac ctgaggaacaccaagtggaacgtgatcatcagggaggacctgctgaggatcgacaacaagacctgcaccctgttcaggaacaaggccgtgcacctggag gtggccaggtacgtgcacgcctacatcaacgacatcgccgaggtgaactcctacttccagctgtaccactacatcatgcagaggatcatcatgaacgagagg tacgagaagtcctccggcaaggtgtccgagtacttcgacgccgtgaacgacgagaagaagtacaacgacaggctgctgaagctgctgtgcgtgccgttcgg ctactgcatcccgaggttcaagaacctgtccatcgaggccctgttcgacaggaacgaggccgccaagttcgacaaggagaagaagaaggtgtccggcaac tcctga。

in the present invention, the method for preparing CasRx mRNA preferably comprises: reacting the system containing the DNA template at 37 ℃ for 6h to obtain CasRx mRNA; the DNA template-containing system preferably comprises per 1600. mu.L: 440 μ L of RNA-free water, 160 μ L of ATP at a concentration of 7.5mM, 160 μ L of UTP at a concentration of 7.5mM, 160 μ L of CTP at a concentration of 7.5mM, 160 μ L of GTP at a concentration of 7.5mM, 160 μ L of Cap analogue at a concentration of 7.5mM, DNA template at a concentration of 150 ng/. mu.L, 160 μ L of 10 XBuffer and 160 μ L of enzyme Mix. In the present invention, when the DNA has no polyA, the resulting CasRx mRNA is added with polyA, and the method of adding polyA to CasRx mRNA is not particularly limited in the present invention and may be added by a person skilled in the art according to a conventional method.

In the present invention, the sgRNA is configured according to the target gene, and the sgRNA directs CasRx protein to bind to the mRNA sequence of the target gene, allowing CasRx to specifically edit (i.e., cleave) the mRNA of the target gene, resulting in down-regulation of the expression of the target gene. When the target gene is preferably PCSK 9gene (Pcsk 9-protein conversion gene subtilisin/kexin type 9Mus musculus (house mouse) Also known as: AI415265, AI747682, FH3, HCHOLA3, Na, Narc1, PC9GeneID:100102), the nucleotide sequence of the sgRNA is preferably shown in SEQ ID No.19 and/or SEQ ID No.20, and when the sgRNA is two sequences, the molar ratio is preferably 1:1, and the specific sequences are as follows:

SEQ IDNo.19：gtgggtgccgtggctgtcacacttgctcgc；

SEQ IDNo.20：gtctgtggaagcgtgtcccatcctcctccg。

in the present invention, when the target gene is preferably Vegfa gene (Vegfa-vascular endothelial growth factor A. Mus musculus (house mouse) Also known as: V, VEGF12, VEGF16, VEGF18, Veg, Vegf, VpfGeneID:22339), the nucleotide sequence of the sgRNA is preferably as shown in SEQ ID No.21 and/or SEQ ID No.22, and when the sgRNA is two sequences, the molar ratio is preferably 1:1, and the specific sequences are as follows:

SEQ IDNo.21：caatcgagaccctggtggacatcttccagg；

SEQ IDNo.22：ggagtgtgtgcccactgaggagtccaacat。

in the present invention, when the target gene is preferably a TYR gene (TYR-TYROSInase Mus musculus (house mouse) Also known as: Oc, Oca1, albino, c, skc3, skc35 GeneID:22173), the nucleotide sequence of the sgRNA is shown in SEQ ID No.23 and/or SEQ ID No.24, and when the sgRNA has two sequences, the molar ratio is preferably 1:1, and the specific sequences are as follows:

SEQ IDNo.23：acctcagttccccttcaaaggggtggatga；

SEQ IDNo.24：taggacctgccagtgctcaggcaacttcat。

in the invention, the mass ratio of the CasRx mRNA to the sgRNA is preferably 5-100: 1, and when the target gene is the PCSK 9gene, the mass ratio of the CasRx mRNA to the sgRNA is preferably 100: 1; when the target gene is Vegfa gene, the mass ratio of the CasRx mRNA to the sgRNA is preferably 5: 1; when the target gene is a tyrosinase gene, the mass ratio of the CasRx mRNA to the sgRNA is preferably 100: 1.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Application of mRNA for coding CasRx in hyperlipidemia

Cellular level: approximately 24 hours after the Huh-7 cells were seeded, the state of the cells in the 6-well plate was observed, and the confluency was about 90%. In a biosafety cabinet, the required volume of 90% DMEM + 10% FBS medium was prepared. The plate was discarded 30min prior to transfection and 1ml of fresh medium (90% MSCBM + 10% FBS) was added per well. Preparing a transfection system: adding 2 mu g of CasRx mRNA and 1 mu g of sgRNA into 200 mu l of opti-MEM, adding only 2 mu g of CasRx mRNA into a negative control group, lightly blowing and uniformly mixing by using a gun head, adding 60 mu l of PEI (the concentration is 1mg/ml), immediately placing on a vortex oscillator for 10 times of oscillation, fully and uniformly mixing 1s each time, and standing for 10 min. The prepared transfection system is directly and evenly dripped into the cultured cells, and then the cells are evenly shaken front and back and left and right, so that the transfection system is evenly distributed on the cells. The medium was changed 6h after transfection, old medium was aspirated off and 2ml fresh medium (90% DMEM + 10% FBS) was changed per well. Total mRNA and total protein were extracted from the cells after 24 hours.

Individual level: the method is characterized in that balb/c mice of 8-week-old males are selected, 2ml of a mixed solution of CasRx mRNA (SEQ ID No. 3-18) and sgRNA is prepared by normal saline in an experimental group, the content of the CasRx mRNA in the mixed solution is 200 mu g, the using amounts of the sgRNA6 and the sgRNA7 are 2 mu g, and the molar ratio of the sgRNA6 to the sgRNA7 is 1:1, injecting 2ml of normal saline into the tail vein of a control group in a rapid tail vein injection mode within 5-7 seconds.

After 24h, primary mouse hepatocytes were isolated by a standard two-step collagenase perfusion method, and the isolated cells were centrifuged in 40% cell isolation (Sigma) at 1000rpm for 10 minutes to obtain hepatocytes. Extracting total mRNA of the liver cells, carrying out reverse transcription and quantitative PCR detection on the abundance of the mRNA of the Pcsk9, extracting total protein of the liver cells, carrying out ELISA and Westernblot detection on the expression level of the protein of the Pcsk 9.

Elisa method:

1. coating: pcsk9 standard was diluted with coatnbuffer to 100ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng in 1ml, total extracted protein was diluted 5/10/20 fold with coatnbuffer and added to the microplate in a volume of 100. mu.l per well, 3 wells repeated for each dilution, the microplate was covered and coated overnight at 4 ℃.

2. Washing the plate: pouring out the coating liquid from the coated 96-well plate to the water absorption paper to force the plate to be fastened until no residue is left in the hole. Preparing an eluent, diluting the eluent by 50x by using deionized water, adding the diluted eluent into a liquid inlet bottle of a plate washing machine, setting a program, setting the volume of each hole of the plate washing machine to be 300 mu l, and repeatedly washing for 4 times.

3. And (3) sealing: the washed plate was rinsed off the solution, a Blocking buffer was added at a volume of 250ul per well, followed by sealing with a sealing plate membrane and sealing at room temperature for 2 h.

4. Washing the plate: and (4) finishing plate washing according to the step (3) by using the closed enzyme label plate.

5. Primary antibody incubation: the anti-Pcsk9 rabbit antibody (1: 2000) was diluted with dilution buffer and added to the washed 96-well plate in a volume of 100. mu.l per well, followed by incubation for 1.5h at room temperature with a sealing plate.

6. Washing the plate: plate washing was completed according to step 3 with the increase in plate washing times to 6.

7. Adding a secondary antibody: diluting HRP-labeled goat anti-rabbit IgG with Diluenbuffer at a dilution ratio of 10000x, adding the diluted antibody into an enzyme label plate according to the volume of 100 mu l per hole, sealing the plate-sealing membrane, and incubating for 1h at room temperature in a dark place.

8. Washing the plate: and (5) finishing plate washing according to the step 8, wherein the plate must be washed clean and the solution is dried.

9. Color development: adding 100. mu.l of TMB buffer, and developing for 20-30 minutes in a dark place, wherein the positive sample develops blue.

10. And (4) terminating: stop buffer 100. mu.l was added and the reading was made on the microplate in 10 minutes, setting the absorption wavelength at 450 nm.

11. And drawing a standard curve according to the pore light absorption value of the standard, wherein the correlation coefficient of linear regression is greater than 0.0995. And calculating the content of Pcsk9 in the sample according to the absorbance of the test sample.

Mice were fasted for 4 hours prior to whole blood collection. Whole blood was then collected at euthanasia. The whole blood was allowed to stand at room temperature for 1 hour, centrifuged at 2000g for 20 minutes, and the serum was transferred to a new tube for further analysis. Serum cholesterol levels were determined using mouse cholesterol detection reagent (Thermo Fisher) according to the instructions.

Pcsk9 sgRNA sequence design:

Pcsk9 sgRNA 1(SEQ ID No.25)：gattagctgactcttccgaataaactcca；

Pcsk9 sgRNA2(SEQ ID No.26)：ggagtagaggcaggcgtcgtcccggaagtt；

Pcsk9 sgRNA3(SEQ ID No.27)：gtgcccttcccttgacagttgagcacacgc；

Pcsk9 sgRNA4(SEQ ID No.28)：ggctgtgcaggctggtgcccttggccacac；

Pcsk9 sgRNA5(SEQ ID No.29)：cagcatcccggccgctgaccacacctgcca；

Pcsk9 sgRNA6(SEQ ID No.19)：gtgggtgccgtggctgtcacacttgctcgc；

Pcsk9 sgRNA7(SEQ ID No.20)：gtctgtggaagcgtgtcccatcctcctccg；

Pcsk9 sgRNA8(SEQ ID No.30)：gtcccatcctcctccggcacgctgttgaag；

Pcsk9 sgRNA9(SEQ ID No.31)：gtgatggtgaccctgccctcaatctcccga；

Pcsk9 sgRNA 10(SEQ ID No.32)：gcaccctggatgctggtatctaagagatac。

the results show that the cellular level Pcsk9 mRNA level of the experimental group is reduced compared with that of the control group, and can be reduced by 90 percent at most (figure 2); meanwhile, the combined use of sgRNA6 and sgRNA7 effectively reduces the mRNA level and protein expression level of Pcsk9 in liver (fig. 3), and the cholesterol level in blood of mouse is nearly 60% (fig. 4), indicating that the high-efficiency silencing of Pcsk9 is successfully realized in mouse liver, and confirming that the CasRx system also has the activity of targeting silencing RNA in adult animals, and influences the expression of glycolipid metabolism related genes. This provides a new approach to the treatment of acquired metabolic diseases.

Example 3

Application of mRNA encoding CasRx in macular degeneration

Cellular level: about 24 hours after the completion of the inoculation of N2a cells, the state of the cells in the 6-well plate was observed, and the degree of confluence was about 90%. In a biosafety cabinet, the required volume of 90% DMEM + 10% FBS medium was prepared. The plate was discarded 30min prior to transfection and 1ml of fresh medium (90% MSCBM + 10% FBS) was added per well. Preparing a transfection system: and adding 2 mu g of CasRx mRNA (SEQ ID No. 3-18) and 1 mu g of sgRNA into 200 mu l of opti-MEM, adding only 2 mu g of CasRx mRNA into a negative control group, gently blowing and uniformly mixing by using a gun head, adding 60 mu l of PEI (with the concentration of 1mg/ml), immediately placing on a vortex oscillator for 10 times of oscillation, fully mixing for 1s each time, and standing for 10 min. The prepared transfection system is directly and evenly dripped into the cultured cells, and then the cells are evenly shaken front and back and left and right, so that the transfection system is evenly distributed on the cells. The medium was changed 6h after transfection, old medium was aspirated off and 2ml fresh medium (90% DMEM + 10% FBS) was changed per well. After 24 hours, total mRNA and total protein of the cells are extracted and subjected to qPCR, ELISA and western blot detection respectively.

Individual level: the experimental subject is an AMD model mouse of 6 weeks old, the concentration of CasRx mRNA in the mixed solution is 5 mug/mug by adopting the mode of subretinal injection, the concentrations of sgRNA5 and sgRNA are both 1 mug/mug, and the total volume is 5 ul.

The eye drops of topicamide phenylephrine for subretinal injection dilate pupils of mice. A drop of sodium hyaluronate was then applied to the cornea for better visualization. The cornea was penetrated under an olympus microscope (olympus, tokyo, japan) using a 30G needle. Next, a Hamilton syringe (32G needle) was inserted through the corneal perforation and the drug was slowly injected. One drop of ofloxacin after injection prevented infection. The degree of macular lesion recovery in mice was then evaluated by measuring the preference of mice for darkness over 1, 3, 5, 7 days. And (3) taking mouse eyeballs at 7 days for protein and RNA extraction, and carrying out qPCR and Westernblot detection.

Vegfa sgRNA sequence design:

Vegfa sgRNA 1(SEQ ID No.33)：gctgtcttgggtgcattggagccttgcctt；

Vegfa sgRNA2(SEQ ID No.34)：ctccaccatgccaagtggtcccaggctgc；

Vegfa sgRNA3(SEQ ID No.35)：ggcagaaggaggagggcagaatcatcacga；

Vegfa sgRNA4(SEQ ID No.36)：gtgaagttcatggatgtctatcagcgcagctac；

Vegfa sgRNA5(SEQ ID No.21)：caatcgagaccctggtggacatcttccagg；

Vegfa sgRNA6(SEQ ID No.37)：cctgatgagatcgagtacatcttcaagcca；

Vegfa sgRNA7(SEQ ID No.38)：cccctgatgcgatgcgggggctgctgcaat；

Vegfa sgRNA8(SEQ ID No.22)：ggagtgtgtgcccactgaggagtccaacat；

Vegfa sgRNA9(SEQ ID No.39)：gaccaaagaaagatagagcaagacaaga；

Vegfa sgRNA 10(SEQ ID No.40)：gtttgtacaagatccgcagacgtgtaaatgt。

the results show that after editing Vegfa mRNA using CasRx and sgRNA, Vegfa mRNA levels were significantly reduced at the cellular level relative to the control group (22%, fig. 5), at the tissue level by 90%, and at the protein expression level (fig. 6). Light-dark preference experiments in AMD mice demonstrated significant recovery of photosensitivity in mice after treatment (figure 7). This example is the potential of the RNA-based CasRx system for the prevention of severe ocular disease-age related macular degeneration (AMD).

Example 4

Application of mRNA for coding CasRx in skin whitening

The process of melanogenesis begins with the oxidation of L-tyrosine to Dopaquinone (DQ) by the key enzyme, Tyrosinase (TYR). The tyrosinase gene family has 3 kinds of genes in total, namely TYR, TYRP1 and TYRP2, wherein TYRP1 and TYRP2 play a catalytic role in controlling the final steps of melanin type production by melanocytes, TYR is a key rate-limiting enzyme in the initial process of melanin synthesis, has at least two activities of tyrosine hydroxylase and dopa oxidase, and is involved in the oxidation and transport of tyrosinase during the melanin synthesis. Tyrosine generates dopa under the action of tyrosinase, and melanin is finally generated through a series of steps.

Cellular level: about 24 hours after the completion of the inoculation of N2a cells, the state of the cells in the 6-well plate was observed, and the degree of confluence was about 90%. In a biosafety cabinet, the required volume of 90% DMEM + 10% FBS medium was prepared. The plate was discarded 30min prior to transfection and 1ml of fresh medium (90% MSCBM + 10% FBS) was added per well. Preparing a transfection system: and adding 2 mu g of CasRx mRNA (SEQ ID No. 3-18) and 1 mu g of sgRNA into 200 mu l of opti-MEM, adding only 2 mu g of CasRx mRNA into a negative control group, gently blowing and uniformly mixing by using a gun head, adding 60 mu l of PEI (with the concentration of 1mg/ml), immediately placing on a vortex oscillator for 10 times of oscillation, fully mixing for 1s each time, and standing for 10 min. The prepared transfection system is directly and evenly dripped into the cultured cells, and then the cells are evenly shaken front and back and left and right, so that the transfection system is evenly distributed on the cells. The medium was changed 6h after transfection, old medium was aspirated off and 2ml fresh medium (90% DMEM + 10% FBS) was changed per well. Total mRNA and total protein were extracted from the cells after 24 hours.

Individual level: the backs of C57 mice were depilated with depilatory cream one day in advance, and the second day, mixed solution of CasRx mRNA prepared with normal saline and sgRNA3+4 for TYR gene was injected into the skin, the total volume was 100. mu.l, the concentration of CasRx mRNA was 2mg/ml, the total concentration of sgRNA3 and sgRNA4 was 20. mu.g/ml, the molar ratio of sgRNA3 and sgRNA4 was 1:1, and the control group was injected with 100. mu.l of normal saline. The degree of accumulation of skin melanin color was observed at 1d, 3d, 5d, and 7 d. And 7d, taking the skin of the injection part of the mouse, extracting RNA and total protein, and respectively carrying out qPCR, ELISA and Westernblot detection.

TYR sgRNA 1(SEQ ID No.41)：agtttccagatctctgatggccattttcct；

TYR sgRNA2(SEQ ID No.42)：ggcaaaagaatgctgcccaccatggatg；

TYR sgRNA3(SEQ ID No.23)：acctcagttccccttcaaaggggtggatga；

TYR sgRNA4(SEQ ID No.24)：taggacctgccagtgctcaggcaacttcat；

TYR sgRNA 5(SEQ ID No.43)：tgtctccgaaaagaataagttcttttcttacc；

TYR sgRNA6(SEQ ID No.44)：tcagtctatgtcatccccacaggcacctat；

TYR sgRNA7(SEQ ID No.45)：tggagagatgcagaaaactgtgacatttg；

TYR sgRNA 8(SEQ ID No.46)：ccatcttcagcagatgtggaattttgtctga；

TYR sgRNA9(SEQ ID No.47)：agcagatccttctcaaagtagcatgcacaa；

TYR sgRNA 10(SEQ ID No.48)：atggtgatttcttcataacatccaaggatct。

The results showed that cellular levels of CasRx/sgRNA significantly down-regulated TYR mRNA levels, with the experimental group having a minimum of 16% of the TYR mRNA levels of the control group (fig. 8). At the level of individual tissues, TYR mRNA decreased to 25% of the control group after regulation by the CasRx system, while TYR protein expression levels decreased significantly (fig. 9). The level of melanin accumulation decreased and mice had low degree of darkening of skin color (fig. 10).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shenzhen Ruiji Biotechnology Limited

<120> CasRx preparation for silencing target gene and application thereof

<160> 48

<170> SIPOSequenceListing 1.0

<210> 1

<211> 967

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly Val Lys Ser

1 5 10 15

Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe Ala Glu Gly

20 25 30

Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser Ile Arg Ser

35 40 45

Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp Lys Asn Ala

50 55 60

Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys Gly Tyr Ala

65 70 75 80

Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln Gln Asp Met

85 90 95

Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly Glu Ser Ala

100 105 110

Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn Ile Leu Asp

115 120 125

Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala Tyr Ala Val

130 135 140

Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe Gly Lys Phe

145 150 155 160

Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu His His Arg

165 170 175

Ala Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala Ile Lys Ala Gln

180 185 190

Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg Leu Gly Tyr Phe

195 200 205

Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr Ile Ile Asn Tyr

210 215 220

Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser Gly Leu Arg His

225 230 235 240

Trp Val Val His Asn Asn Glu Glu Glu Ser Arg Ile Ser Arg Thr Trp

245 250 255

Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr Ile Ser Thr Leu

260 265 270

Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr Asn Ser Phe Ser

275 280 285

Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu Thr Leu Gly Ile

290 295 300

Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe Ser Ile Met Lys

305 310 315 320

Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu Arg Glu Val Met

325 330 335

Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn His Lys Val Phe

340 345 350

Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp Phe Val Ile Tyr

355 360 365

Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala Ala Asn Lys Ser

370 375 380

Leu Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp Ile Phe Val Ile

385 390 395 400

Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp Ala Leu Tyr Tyr

405 410 415

Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn Ile Met His Asn

420 425 430

Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr Lys Lys Lys Asp

435 440 445

Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg Asp Val Ser Ala

450 455 460

Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu Asp Gly Lys Glu

465 470 475 480

Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp Asn Ile Gln

485 490 495

Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn Ala Lys Phe Val

500 505 510

Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile Ala Asp Glu Leu

515 520 525

Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro Ile Ala Asp Ala

530 535 540

Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu Gly Thr Asn Leu

545 550 555 560

Ser Tyr Asp Glu Leu Lys Ala Leu Ala Asp Thr Phe Ser Leu Asp Glu

565 570 575

Asn Gly Asn Lys Leu Lys Lys Gly Lys His Gly Met Arg Asn Phe Ile

580 585 590

Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His Tyr Leu Ile Arg Tyr

595 600 605

Gly Asp Pro Ala His Leu His Glu Ile Ala Lys Asn Glu Ala Val Val

610 615 620

Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln Lys Lys Gln Gly Gln

625 630 635 640

Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu Thr Cys Ile Gly Lys

645 650 655

Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp Ala Leu Thr Lys Ile

660 665 670

Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys Lys Arg Ser Val Ile

675 680 685

Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu Lys Phe Lys Lys Ile

690 695 700

Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile Leu Lys Asn Ile Val

705 710 715 720

Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe His Cys Val Glu Arg Asp

725 730 735

Ala Gln Leu Tyr Lys Glu Lys Gly Tyr Asp Ile Asn Leu Lys Lys Leu

740 745 750

Glu Glu Lys Gly Phe Ser Ser Val Thr Lys Leu Cys Ala Gly Ile Asp

755 760 765

Glu Thr Ala Pro Asp Lys Arg Lys Asp Val Glu Lys Glu Met Ala Glu

770 775 780

Arg Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser Ala Asn Pro Lys Leu

785 790 795 800

Tyr Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys Lys Ala Glu Glu Phe

805 810 815

Thr Arg Gln Ile Asn Arg Glu Lys Ala Lys Thr Ala Leu Asn Ala Tyr

820 825 830

Leu Arg Asn Thr Lys Trp Asn Val Ile Ile Arg Glu Asp Leu Leu Arg

835 840 845

Ile Asp Asn Lys Thr Cys Thr Leu Phe Arg Asn Lys Ala Val His Leu

850 855 860

Glu Val Ala Arg Tyr Val His Ala Tyr Ile Asn Asp Ile Ala Glu Val

865 870 875 880

Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met Gln Arg Ile Ile Met

885 890 895

Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val Ser Glu Tyr Phe Asp

900 905 910

Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg Leu Leu Lys Leu Leu

915 920 925

Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe Lys Asn Leu Ser Ile

930 935 940

Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys Phe Asp Lys Glu Lys

945 950 955 960

Lys Lys Val Ser Gly Asn Ser

965

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cccaagaaaa agagaaaggt g 21

<210> 3

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgatcgaaa agaaaaagag cttcgcgaag ggcatgggcg tgaagagcac cctggtgagc 60

ggcagcaagg tgtacatgac caccttcgcc gagggcagcg acgctagact ggagaagatc 120

gtggagggcg acagcatcag atccgtgaac gagggcgagg ccttcagcgc cgagatggcc 180

gacaagaacg ccggctacaa gatcggcaac gccaagttca gccatcccaa gggctacgcc 240

gtggtggcca acaaccccct gtacaccggc cccgtgcagc aagacatgct gggcctgaag 300

gagaccctgg agaagagata cttcggcgag agcgccgacg gcaacgacaa catctgcatc 360

caagtgatcc acaacatcct ggacatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat cagcggcctg gacaaggaca tcatcggctt cggcaagttc 480

agcaccgtgt acacctacga cgagttcaaa gatcccgagc accacagagc cgccttcaac 540

aacaacgaca agctgatcaa cgccatcaag gctcagtatg acgagtttga taatttcctg 600

gacaacccta gactgggcta cttcggccaa gccttcttca gcaaggaggg cagaaactac 660

atcatcaact acggcaacga gtgctacgac atcctggccc tgctgagcgg cctgagacac 720

tgggtggtgc acaacaacga ggaggagagc agaatcagca gaacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatcagc accctgaact acctgtacga cagaatcacc 840

aacgagctga ccaacagctt cagcaagaac agcgccgcca acgtgaacta catcgccgag 900

accctgggca tcaaccccgc cgagttcgcc gagcagtact tcagattcag catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgagag aggtgatgct ggacagaaag 1020

gacatgagcg agatcagaaa gaaccacaag gtgttcgata gcataaggac caaggtgtac 1080

accatgatgg acttcgtgat ctacagatac tacatcgagg aggacgccaa ggtggccgcc 1140

gccaacaaga gcctgcccga caacgagaag agcctcagcg agaaggacat cttcgtgatc 1200

aacctgagag gcagcttcaa cgacgatcag aaggacgcac tgtattacga cgaggccaac 1260

agaatctgga gaaagctgga gaacatcatg cacaacatca aggagttcag aggcaacaag 1320

acaagagagt acaagaagaa ggatgcccct agactgccta gaatcctgcc cgccggcaga 1380

gacgtgagcg ccttcagcaa gctgatgtac gccctgacca tgttcctgga cggcaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagtttgaca acattcagag cttcctgaag 1500

gtgatgcccc tgatcggcgt gaacgccaag tttgtggagg agtacgcctt cttcaaggac 1560

agcgccaaga tcgccgacga gctgagactg atcaagagtt tcgcgcggat gggcgagccc 1620

atcgccgacg ctagaagagc catgtacatc gacgccatca gaatcctggg caccaacctg 1680

agctacgacg agctgaaggc cctggccgac accttcagcc tggacgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgagaaac ttcatcatca acaacgtgat cagcaacaag 1800

agattccact acctgatcag atacggcgac cccgcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggcaga atcgccgaca ttcagaagaa gcaagggcag 1920

aacggcaaga atcagatcga cagatactac gagacctgca tcggcaagga caagggcaag 1980

agcgttagcg aaaaggttga cgccctgaca aagatcatca ccggcatgaa ctacgatcag 2040

ttcgacaaga agagaagcgt gatcgaggac accggcagag agaacgccga gagagagaag 2100

ttcaagaaga tcatcagcct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ctagatacgt tatcggcttc cactgcgtgg agagagacgc tcagctgtac 2220

aaagagaagg gttacgacat caacctgaag aagctggagg agaagggctt tagcagcgtg 2280

accaagctgt gcgccggcat cgacgagacc gcccccgaca agagaaagga cgtggagaag 2340

gagatggccg agagagccaa ggagagcatc gacagcctgg agagcgccaa ccccaagctg 2400

tacgccaact acatcaagta cagcgacgag aagaaggccg aggagttcac aagacagatc 2460

aacagagaga aggccaagac cgccctgaac gcctacctga gaaacaccaa gtggaacgtg 2520

atcatcagag aggacctgct gagaatcgac aacaagacct gcaccctgtt cagaaacaag 2580

gccgtgcacc tggaggtggc tagatacgtt catgcctaca tcaacgacat cgccgaggtg 2640

aacagctact ttcagctgta ccactacatc atgcagagaa tcatcatgaa cgagagatac 2700

gaaaagagca gcggcaaggt gagcgagtac ttcgacgccg tgaacgatga gaagaaatat 2760

aacgacagac tgctgaagct gctgtgcgtg cccttcggct actgcatccc tagattcaag 2820

aacctgagca tcgaggccct gttcgacaga aacgaggccg ccaagttcga taaggaaaaa 2880

aagaaagttt cgggcaacag ctaa 2904

<210> 4

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgattgaaa agaagaagtc ttttgctaag ggaatgggag ttaagtctac tcttgtttct 60

ggatctaagg tttatatgac tacttttgct gaaggatctg atgctagact tgaaaagatt 120

gttgaaggag attctattag atctgttaat gaaggagaag ctttttctgc tgaaatggct 180

gataagaatg ctggatataa gattggaaat gctaagtttt ctcatcctaa gggatatgct 240

gttgttgcta ataatcctct ttatactgga cctgttcaac aagatatgct tggacttaag 300

gaaactcttg aaaagagata ttttggagaa tctgctgatg gaaatgataa tatttgtatt 360

caagttattc ataatattct tgatattgaa aagattcttg ctgaatatat tactaatgct 420

gcttatgctg ttaataatat ttctggactt gataaggata ttattggatt tggaaagttt 480

tctactgttt atacttatga tgaatttaag gatcctgaac atcatagagc tgcttttaat 540

aataatgata agcttattaa tgctattaag gctcaatatg atgaatttga taattttctt 600

gataatccta gacttggata ttttggacaa gctttttttt ctaaggaagg aagaaattat 660

attattaatt atggaaatga atgttatgat attcttgctc ttctttctgg acttagacat 720

tgggttgttc ataataatga agaagaatct agaatttcta gaacttggct ttataatctt 780

gataagaatc ttgataatga atatatttct actcttaatt atctttatga tagaattact 840

aatgaactta ctaattcttt ttctaagaat tctgctgcta atgttaatta tattgctgaa 900

actcttggaa ttaatcctgc tgaatttgct gaacaatatt ttagattttc tattatgaag 960

gaacaaaaga atcttggatt taatattact aagcttagag aagttatgct tgatagaaag 1020

gatatgtctg aaattagaaa gaatcataag gtttttgatt ctattagaac taaggtttat 1080

actatgatgg attttgttat ttatagatat tatattgaag aagatgctaa ggttgctgct 1140

gctaataagt ctcttcctga taatgaaaag tctctttctg aaaaggatat ttttgttatt 1200

aatcttagag gatcttttaa tgatgatcaa aaggatgctc tttattatga tgaagctaat 1260

agaatttgga gaaagcttga aaatattatg cataatatta aggaatttag aggaaataag 1320

actagagaat ataagaagaa ggatgctcct agacttccta gaattcttcc tgctggaaga 1380

gatgtttctg ctttttctaa gcttatgtat gctcttacta tgtttcttga tggaaaggaa 1440

attaatgatc ttcttactac tcttattaat aagtttgata atattcaatc ttttcttaag 1500

gttatgcctc ttattggagt taatgctaag tttgttgaag aatatgcttt ttttaaggat 1560

tctgctaaga ttgctgatga acttagactt attaagtctt ttgctagaat gggagaacct 1620

attgctgatg ctagaagagc tatgtatatt gatgctatta gaattcttgg aactaatctt 1680

tcttatgatg aacttaaggc tcttgctgat actttttctc ttgatgaaaa tggaaataag 1740

cttaagaagg gaaagcatgg aatgagaaat tttattatta ataatgttat ttctaataag 1800

agatttcatt atcttattag atatggagat cctgctcatc ttcatgaaat tgctaagaat 1860

gaagctgttg ttaagtttgt tcttggaaga attgctgata ttcaaaagaa gcaaggacaa 1920

aatggaaaga atcaaattga tagatattat gaaacttgta ttggaaagga taagggaaag 1980

tctgtttctg aaaaggttga tgctcttact aagattatta ctggaatgaa ttatgatcaa 2040

tttgataaga agagatctgt tattgaagat actggaagag aaaatgctga aagagaaaag 2100

tttaagaaga ttatttctct ttatcttact gttatttatc atattcttaa gaatattgtt 2160

aatattaatg ctagatatgt tattggattt cattgtgttg aaagagatgc tcaactttat 2220

aaggaaaagg gatatgatat taatcttaag aagcttgaag aaaagggatt ttcttctgtt 2280

actaagcttt gtgctggaat tgatgaaact gctcctgata agagaaagga tgttgaaaag 2340

gaaatggctg aaagagctaa ggaatctatt gattctcttg aatctgctaa tcctaagctt 2400

tatgctaatt atattaagta ttctgatgaa aagaaggctg aagaatttac tagacaaatt 2460

aatagagaaa aggctaagac tgctcttaat gcttatctta gaaatactaa gtggaatgtt 2520

attattagag aagatcttct tagaattgat aataagactt gtactctttt tagaaataag 2580

gctgttcatc ttgaagttgc tagatatgtt catgcttata ttaatgatat tgctgaagtt 2640

aattcttatt ttcaacttta tcattatatt atgcaaagaa ttattatgaa tgaaagatat 2700

gaaaagtctt ctggaaaggt ttctgaatat tttgatgctg ttaatgatga aaagaagtat 2760

aatgatagac ttcttaagct tctttgtgtt ccttttggat attgtattcc tagatttaag 2820

aatctttcta ttgaagctct ttttgataga aatgaagctg ctaagtttga taaggaaaag 2880

aagaaggttt ctggaaattc ttga 2904

<210> 5

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgattgaaa aaaaaaaatc attcgctaaa ggaatgggag ttaaatcaac acttgtttca 60

ggatcaaaag tttatatgac aacattcgct gaaggatcag atgctagact tgaaaaaatt 120

gttgaaggag attcaattag atcagttaat gaaggagaag ctttctcagc tgaaatggct 180

gataaaaatg ctggatataa aattggaaat gctaaattct cacatccaaa aggatatgct 240

gttgttgcta ataatccact ttatacagga ccagttcaac aagatatgct tggacttaaa 300

gaaacacttg aaaaaagata tttcggagaa tcagctgatg gaaatgataa tatttgtatt 360

caagttattc ataatattct tgatattgaa aaaattcttg ctgaatatat tacaaatgct 420

gcttatgctg ttaataatat ttcaggactt gataaagata ttattggatt cggaaaattc 480

tcaacagttt atacatatga tgaattcaaa gatccagaac atcatagagc tgctttcaat 540

aataatgata aacttattaa tgctattaaa gctcaatatg atgaattcga taatttcctt 600

gataatccaa gacttggata tttcggacaa gctttcttct caaaagaagg aagaaattat 660

attattaatt atggaaatga atgttatgat attcttgctc ttctttcagg acttagacat 720

tgggttgttc ataataatga agaagaatca agaatttcaa gaacatggct ttataatctt 780

gataaaaatc ttgataatga atatatttca acacttaatt atctttatga tagaattaca 840

aatgaactta caaattcatt ctcaaaaaat tcagctgcta atgttaatta tattgctgaa 900

acacttggaa ttaatccagc tgaattcgct gaacaatatt tcagattctc aattatgaaa 960

gaacaaaaaa atcttggatt caatattaca aaacttagag aagttatgct tgatagaaaa 1020

gatatgtcag aaattagaaa aaatcataaa gttttcgatt caattagaac aaaagtttat 1080

acaatgatgg atttcgttat ttatagatat tatattgaag aagatgctaa agttgctgct 1140

gctaataaat cacttccaga taatgaaaaa tcactttcag aaaaagatat tttcgttatt 1200

aatcttagag gatcattcaa tgatgatcaa aaagatgctc tttattatga tgaagctaat 1260

agaatttgga gaaaacttga aaatattatg cataatatta aagaattcag aggaaataaa 1320

acaagagaat ataaaaaaaa agatgctcca agacttccaa gaattcttcc agctggaaga 1380

gatgtttcag ctttctcaaa acttatgtat gctcttacaa tgttccttga tggaaaagaa 1440

attaatgatc ttcttacaac acttattaat aaattcgata atattcaatc attccttaaa 1500

gttatgccac ttattggagt taatgctaaa ttcgttgaag aatatgcttt cttcaaagat 1560

tcagctaaaa ttgctgatga acttagactt attaaatcat tcgctagaat gggagaacca 1620

attgctgatg ctagaagagc tatgtatatt gatgctatta gaattcttgg aacaaatctt 1680

tcatatgatg aacttaaagc tcttgctgat acattctcac ttgatgaaaa tggaaataaa 1740

cttaaaaaag gaaaacatgg aatgagaaat ttcattatta ataatgttat ttcaaataaa 1800

agattccatt atcttattag atatggagat ccagctcatc ttcatgaaat tgctaaaaat 1860

gaagctgttg ttaaattcgt tcttggaaga attgctgata ttcaaaaaaa acaaggacaa 1920

aatggaaaaa atcaaattga tagatattat gaaacatgta ttggaaaaga taaaggaaaa 1980

tcagtttcag aaaaagttga tgctcttaca aaaattatta caggaatgaa ttatgatcaa 2040

ttcgataaaa aaagatcagt tattgaagat acaggaagag aaaatgctga aagagaaaaa 2100

ttcaaaaaaa ttatttcact ttatcttaca gttatttatc atattcttaa aaatattgtt 2160

aatattaatg ctagatatgt tattggattc cattgtgttg aaagagatgc tcaactttat 2220

aaagaaaaag gatatgatat taatcttaaa aaacttgaag aaaaaggatt ctcatcagtt 2280

acaaaacttt gtgctggaat tgatgaaaca gctccagata aaagaaaaga tgttgaaaaa 2340

gaaatggctg aaagagctaa agaatcaatt gattcacttg aatcagctaa tccaaaactt 2400

tatgctaatt atattaaata ttcagatgaa aaaaaagctg aagaattcac aagacaaatt 2460

aatagagaaa aagctaaaac agctcttaat gcttatctta gaaatacaaa atggaatgtt 2520

attattagag aagatcttct tagaattgat aataaaacat gtacactttt cagaaataaa 2580

gctgttcatc ttgaagttgc tagatatgtt catgcttata ttaatgatat tgctgaagtt 2640

aattcatatt tccaacttta tcattatatt atgcaaagaa ttattatgaa tgaaagatat 2700

gaaaaatcat caggaaaagt ttcagaatat ttcgatgctg ttaatgatga aaaaaaatat 2760

aatgatagac ttcttaaact tctttgtgtt ccattcggat attgtattcc aagattcaaa 2820

aatctttcaa ttgaagctct tttcgataga aatgaagctg ctaaattcga taaagaaaaa 2880

aaaaaagttt caggaaattc atga 2904

<210> 6

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgatcgaga agaagaagtc cttcgccaag ggcatgggcg tgaagtccac cctggtgtcc 60

ggctccaagg tgtacatgac caccttcgcc gagggctccg acgccaggct ggagaagatc 120

gtggagggcg actccatcag gtccgtgaac gagggcgagg ccttctccgc cgagatggcc 180

gacaagaacg ccggctacaa gatcggcaac gccaagttct cccaccccaa gggctacgcc 240

gtggtggcca acaaccccct gtacaccggc cccgtgcagc aggacatgct gggcctgaag 300

gagaccctgg agaagaggta cttcggcgag tccgccgacg gcaacgacaa catctgcatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat ctccggcctg gacaaggaca tcatcggctt cggcaagttc 480

tccaccgtgt acacctacga cgagttcaag gaccccgagc accacagggc cgccttcaac 540

aacaacgaca agctgatcaa cgccatcaag gcccagtacg acgagttcga caacttcctg 600

gacaacccca ggctgggcta cttcggccag gccttcttct ccaaggaggg caggaactac 660

atcatcaact acggcaacga gtgctacgac atcctggccc tgctgtccgg cctgaggcac 720

tgggtggtgc acaacaacga ggaggagtcc aggatctcca ggacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatctcc accctgaact acctgtacga caggatcacc 840

aacgagctga ccaactcctt ctccaagaac tccgccgcca acgtgaacta catcgccgag 900

accctgggca tcaaccccgc cgagttcgcc gagcagtact tcaggttctc catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgaggg aggtgatgct ggacaggaag 1020

gacatgtccg agatcaggaa gaaccacaag gtgttcgact ccatcaggac caaggtgtac 1080

accatgatgg acttcgtgat ctacaggtac tacatcgagg aggacgccaa ggtggccgcc 1140

gccaacaagt ccctgcccga caacgagaag tccctgtccg agaaggacat cttcgtgatc 1200

aacctgaggg gctccttcaa cgacgaccag aaggacgccc tgtactacga cgaggccaac 1260

aggatctgga ggaagctgga gaacatcatg cacaacatca aggagttcag gggcaacaag 1320

accagggagt acaagaagaa ggacgccccc aggctgccca ggatcctgcc cgccggcagg 1380

gacgtgtccg ccttctccaa gctgatgtac gccctgacca tgttcctgga cggcaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagttcgaca acatccagtc cttcctgaag 1500

gtgatgcccc tgatcggcgt gaacgccaag ttcgtggagg agtacgcctt cttcaaggac 1560

tccgccaaga tcgccgacga gctgaggctg atcaagtcct tcgccaggat gggcgagccc 1620

atcgccgacg ccaggagggc catgtacatc gacgccatca ggatcctggg caccaacctg 1680

tcctacgacg agctgaaggc cctggccgac accttctccc tggacgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgaggaac ttcatcatca acaacgtgat ctccaacaag 1800

aggttccact acctgatcag gtacggcgac cccgcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggcagg atcgccgaca tccagaagaa gcagggccag 1920

aacggcaaga accagatcga caggtactac gagacctgca tcggcaagga caagggcaag 1980

tccgtgtccg agaaggtgga cgccctgacc aagatcatca ccggcatgaa ctacgaccag 2040

ttcgacaaga agaggtccgt gatcgaggac accggcaggg agaacgccga gagggagaag 2100

ttcaagaaga tcatctccct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ccaggtacgt gatcggcttc cactgcgtgg agagggacgc ccagctgtac 2220

aaggagaagg gctacgacat caacctgaag aagctggagg agaagggctt ctcctccgtg 2280

accaagctgt gcgccggcat cgacgagacc gcccccgaca agaggaagga cgtggagaag 2340

gagatggccg agagggccaa ggagtccatc gactccctgg agtccgccaa ccccaagctg 2400

tacgccaact acatcaagta ctccgacgag aagaaggccg aggagttcac caggcagatc 2460

aacagggaga aggccaagac cgccctgaac gcctacctga ggaacaccaa gtggaacgtg 2520

atcatcaggg aggacctgct gaggatcgac aacaagacct gcaccctgtt caggaacaag 2580

gccgtgcacc tggaggtggc caggtacgtg cacgcctaca tcaacgacat cgccgaggtg 2640

aactcctact tccagctgta ccactacatc atgcagagga tcatcatgaa cgagaggtac 2700

gagaagtcct ccggcaaggt gtccgagtac ttcgacgccg tgaacgacga gaagaagtac 2760

aacgacaggc tgctgaagct gctgtgcgtg cccttcggct actgcatccc caggttcaag 2820

aacctgtcca tcgaggccct gttcgacagg aacgaggccg ccaagttcga caaggagaag 2880

aagaaggtgt ccggcaactc ctga 2904

<210> 7

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgatcgaga agaagaagag cttcgctaag ggaatgggag tgaagagcac actggtgagc 60

ggaagcaagg tgtacatgac aacattcgct gagggaagcg acgctagact ggagaagatc 120

gtggagggag acagcatcag aagcgtgaac gagggagagg ctttcagcgc tgagatggct 180

gacaagaacg ctggatacaa gatcggaaac gctaagttca gccaccctaa gggatacgct 240

gtggtggcta acaaccctct gtacacagga cctgtgcagc aggacatgct gggactgaag 300

gagacactgg agaagagata cttcggagag agcgctgacg gaaacgacaa catctgtatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ctgagtacat cacaaacgct 420

gcttacgctg tgaacaacat cagcggactg gacaaggaca tcatcggatt cggaaagttc 480

agcacagtgt acacatacga cgagttcaag gaccctgagc accacagagc tgctttcaac 540

aacaacgaca agctgatcaa cgctatcaag gctcagtacg acgagttcga caacttcctg 600

gacaacccta gactgggata cttcggacag gctttcttca gcaaggaggg aagaaactac 660

atcatcaact acggaaacga gtgttacgac atcctggctc tgctgagcgg actgagacac 720

tgggtggtgc acaacaacga ggaggagagc agaatcagca gaacatggct gtacaacctg 780

gacaagaacc tggacaacga gtacatcagc acactgaact acctgtacga cagaatcaca 840

aacgagctga caaacagctt cagcaagaac agcgctgcta acgtgaacta catcgctgag 900

acactgggaa tcaaccctgc tgagttcgct gagcagtact tcagattcag catcatgaag 960

gagcagaaga acctgggatt caacatcaca aagctgagag aggtgatgct ggacagaaag 1020

gacatgagcg agatcagaaa gaaccacaag gtgttcgaca gcatcagaac aaaggtgtac 1080

acaatgatgg acttcgtgat ctacagatac tacatcgagg aggacgctaa ggtggctgct 1140

gctaacaaga gcctgcctga caacgagaag agcctgagcg agaaggacat cttcgtgatc 1200

aacctgagag gaagcttcaa cgacgaccag aaggacgctc tgtactacga cgaggctaac 1260

agaatctgga gaaagctgga gaacatcatg cacaacatca aggagttcag aggaaacaag 1320

acaagagagt acaagaagaa ggacgctcct agactgccta gaatcctgcc tgctggaaga 1380

gacgtgagcg ctttcagcaa gctgatgtac gctctgacaa tgttcctgga cggaaaggag 1440

atcaacgacc tgctgacaac actgatcaac aagttcgaca acatccagag cttcctgaag 1500

gtgatgcctc tgatcggagt gaacgctaag ttcgtggagg agtacgcttt cttcaaggac 1560

agcgctaaga tcgctgacga gctgagactg atcaagagct tcgctagaat gggagagcct 1620

atcgctgacg ctagaagagc tatgtacatc gacgctatca gaatcctggg aacaaacctg 1680

agctacgacg agctgaaggc tctggctgac acattcagcc tggacgagaa cggaaacaag 1740

ctgaagaagg gaaagcacgg aatgagaaac ttcatcatca acaacgtgat cagcaacaag 1800

agattccact acctgatcag atacggagac cctgctcacc tgcacgagat cgctaagaac 1860

gaggctgtgg tgaagttcgt gctgggaaga atcgctgaca tccagaagaa gcagggacag 1920

aacggaaaga accagatcga cagatactac gagacatgta tcggaaagga caagggaaag 1980

agcgtgagcg agaaggtgga cgctctgaca aagatcatca caggaatgaa ctacgaccag 2040

ttcgacaaga agagaagcgt gatcgaggac acaggaagag agaacgctga gagagagaag 2100

ttcaagaaga tcatcagcct gtacctgaca gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ctagatacgt gatcggattc cactgtgtgg agagagacgc tcagctgtac 2220

aaggagaagg gatacgacat caacctgaag aagctggagg agaagggatt cagcagcgtg 2280

acaaagctgt gtgctggaat cgacgagaca gctcctgaca agagaaagga cgtggagaag 2340

gagatggctg agagagctaa ggagagcatc gacagcctgg agagcgctaa ccctaagctg 2400

tacgctaact acatcaagta cagcgacgag aagaaggctg aggagttcac aagacagatc 2460

aacagagaga aggctaagac agctctgaac gcttacctga gaaacacaaa gtggaacgtg 2520

atcatcagag aggacctgct gagaatcgac aacaagacat gtacactgtt cagaaacaag 2580

gctgtgcacc tggaggtggc tagatacgtg cacgcttaca tcaacgacat cgctgaggtg 2640

aacagctact tccagctgta ccactacatc atgcagagaa tcatcatgaa cgagagatac 2700

gagaagagca gcggaaaggt gagcgagtac ttcgacgctg tgaacgacga gaagaagtac 2760

aacgacagac tgctgaagct gctgtgtgtg cctttcggat actgtatccc tagattcaag 2820

aacctgagca tcgaggctct gttcgacaga aacgaggctg ctaagttcga caaggagaag 2880

aagaaggtga gcggaaacag ctga 2904

<210> 8

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgatcgaga agaagaagag cttcgccaag ggcatgggcg tgaagagcac cctggtgagc 60

ggcagcaagg tgtacatgac caccttcgcc gagggcagcg atgcccgcct ggagaagatc 120

gtggagggcg atagcatccg cagcgtgaac gagggcgagg ccttcagcgc cgagatggcc 180

gataagaacg ccggctacaa gatcggcaac gccaagttca gccaccccaa gggctacgcc 240

gtggtggcca acaaccccct gtacaccggc cccgtgcagc aggatatgct gggcctgaag 300

gagaccctgg agaagcgcta cttcggcgag agcgccgatg gcaacgataa catctgcatc 360

caggtgatcc acaacatcct ggatatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat cagcggcctg gataaggata tcatcggctt cggcaagttc 480

agcaccgtgt acacctacga tgagttcaag gatcccgagc accaccgcgc cgccttcaac 540

aacaacgata agctgatcaa cgccatcaag gcccagtacg atgagttcga taacttcctg 600

gataaccccc gcctgggcta cttcggccag gccttcttca gcaaggaggg ccgcaactac 660

atcatcaact acggcaacga gtgctacgat atcctggccc tgctgagcgg cctgcgccac 720

tgggtggtgc acaacaacga ggaggagagc cgcatcagcc gcacctggct gtacaacctg 780

gataagaacc tggataacga gtacatcagc accctgaact acctgtacga tcgcatcacc 840

aacgagctga ccaacagctt cagcaagaac agcgccgcca acgtgaacta catcgccgag 900

accctgggca tcaaccccgc cgagttcgcc gagcagtact tccgcttcag catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgcgcg aggtgatgct ggatcgcaag 1020

gatatgagcg agatccgcaa gaaccacaag gtgttcgata gcatccgcac caaggtgtac 1080

accatgatgg atttcgtgat ctaccgctac tacatcgagg aggatgccaa ggtggccgcc 1140

gccaacaaga gcctgcccga taacgagaag agcctgagcg agaaggatat cttcgtgatc 1200

aacctgcgcg gcagcttcaa cgatgatcag aaggatgccc tgtactacga tgaggccaac 1260

cgcatctggc gcaagctgga gaacatcatg cacaacatca aggagttccg cggcaacaag 1320

acccgcgagt acaagaagaa ggatgccccc cgcctgcccc gcatcctgcc cgccggccgc 1380

gatgtgagcg ccttcagcaa gctgatgtac gccctgacca tgttcctgga tggcaaggag 1440

atcaacgatc tgctgaccac cctgatcaac aagttcgata acatccagag cttcctgaag 1500

gtgatgcccc tgatcggcgt gaacgccaag ttcgtggagg agtacgcctt cttcaaggat 1560

agcgccaaga tcgccgatga gctgcgcctg atcaagagct tcgcccgcat gggcgagccc 1620

atcgccgatg cccgccgcgc catgtacatc gatgccatcc gcatcctggg caccaacctg 1680

agctacgatg agctgaaggc cctggccgat accttcagcc tggatgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgcgcaac ttcatcatca acaacgtgat cagcaacaag 1800

cgcttccact acctgatccg ctacggcgat cccgcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggccgc atcgccgata tccagaagaa gcagggccag 1920

aacggcaaga accagatcga tcgctactac gagacctgca tcggcaagga taagggcaag 1980

agcgtgagcg agaaggtgga tgccctgacc aagatcatca ccggcatgaa ctacgatcag 2040

ttcgataaga agcgcagcgt gatcgaggat accggccgcg agaacgccga gcgcgagaag 2100

ttcaagaaga tcatcagcct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg cccgctacgt gatcggcttc cactgcgtgg agcgcgatgc ccagctgtac 2220

aaggagaagg gctacgatat caacctgaag aagctggagg agaagggctt cagcagcgtg 2280

accaagctgt gcgccggcat cgatgagacc gcccccgata agcgcaagga tgtggagaag 2340

gagatggccg agcgcgccaa ggagagcatc gatagcctgg agagcgccaa ccccaagctg 2400

tacgccaact acatcaagta cagcgatgag aagaaggccg aggagttcac ccgccagatc 2460

aaccgcgaga aggccaagac cgccctgaac gcctacctgc gcaacaccaa gtggaacgtg 2520

atcatccgcg aggatctgct gcgcatcgat aacaagacct gcaccctgtt ccgcaacaag 2580

gccgtgcacc tggaggtggc ccgctacgtg cacgcctaca tcaacgatat cgccgaggtg 2640

aacagctact tccagctgta ccactacatc atgcagcgca tcatcatgaa cgagcgctac 2700

gagaagagca gcggcaaggt gagcgagtac ttcgatgccg tgaacgatga gaagaagtac 2760

aacgatcgcc tgctgaagct gctgtgcgtg cccttcggct actgcatccc ccgcttcaag 2820

aacctgagca tcgaggccct gttcgatcgc aacgaggccg ccaagttcga taaggagaag 2880

aagaaggtga gcggcaacag ctga 2904

<210> 9

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgattgaaa aaaaaaaaag ctttgcgaaa ggcatgggcg tgaaaagcac cctggtgagc 60

ggcagcaaag tgtatatgac cacctttgcg gaaggcagcg atgcgcgcct ggaaaaaatt 120

gtggaaggcg atagcattcg cagcgtgaac gaaggcgaag cgtttagcgc ggaaatggcg 180

gataaaaacg cgggctataa aattggcaac gcgaaattta gccatccgaa aggctatgcg 240

gtggtggcga acaacccgct gtataccggc ccggtgcagc aggatatgct gggcctgaaa 300

gaaaccctgg aaaaacgcta ttttggcgaa agcgcggatg gcaacgataa catttgcatt 360

caggtgattc ataacattct ggatattgaa aaaattctgg cggaatatat taccaacgcg 420

gcgtatgcgg tgaacaacat tagcggcctg gataaagata ttattggctt tggcaaattt 480

agcaccgtgt atacctatga tgaatttaaa gatccggaac atcatcgcgc ggcgtttaac 540

aacaacgata aactgattaa cgcgattaaa gcgcagtatg atgaatttga taactttctg 600

gataacccgc gcctgggcta ttttggccag gcgtttttta gcaaagaagg ccgcaactat 660

attattaact atggcaacga atgctatgat attctggcgc tgctgagcgg cctgcgccat 720

tgggtggtgc ataacaacga agaagaaagc cgcattagcc gcacctggct gtataacctg 780

gataaaaacc tggataacga atatattagc accctgaact atctgtatga tcgcattacc 840

aacgaactga ccaacagctt tagcaaaaac agcgcggcga acgtgaacta tattgcggaa 900

accctgggca ttaacccggc ggaatttgcg gaacagtatt ttcgctttag cattatgaaa 960

gaacagaaaa acctgggctt taacattacc aaactgcgcg aagtgatgct ggatcgcaaa 1020

gatatgagcg aaattcgcaa aaaccataaa gtgtttgata gcattcgcac caaagtgtat 1080

accatgatgg attttgtgat ttatcgctat tatattgaag aagatgcgaa agtggcggcg 1140

gcgaacaaaa gcctgccgga taacgaaaaa agcctgagcg aaaaagatat ttttgtgatt 1200

aacctgcgcg gcagctttaa cgatgatcag aaagatgcgc tgtattatga tgaagcgaac 1260

cgcatttggc gcaaactgga aaacattatg cataacatta aagaatttcg cggcaacaaa 1320

acccgcgaat ataaaaaaaa agatgcgccg cgcctgccgc gcattctgcc ggcgggccgc 1380

gatgtgagcg cgtttagcaa actgatgtat gcgctgacca tgtttctgga tggcaaagaa 1440

attaacgatc tgctgaccac cctgattaac aaatttgata acattcagag ctttctgaaa 1500

gtgatgccgc tgattggcgt gaacgcgaaa tttgtggaag aatatgcgtt ttttaaagat 1560

agcgcgaaaa ttgcggatga actgcgcctg attaaaagct ttgcgcgcat gggcgaaccg 1620

attgcggatg cgcgccgcgc gatgtatatt gatgcgattc gcattctggg caccaacctg 1680

agctatgatg aactgaaagc gctggcggat acctttagcc tggatgaaaa cggcaacaaa 1740

ctgaaaaaag gcaaacatgg catgcgcaac tttattatta acaacgtgat tagcaacaaa 1800

cgctttcatt atctgattcg ctatggcgat ccggcgcatc tgcatgaaat tgcgaaaaac 1860

gaagcggtgg tgaaatttgt gctgggccgc attgcggata ttcagaaaaa acagggccag 1920

aacggcaaaa accagattga tcgctattat gaaacctgca ttggcaaaga taaaggcaaa 1980

agcgtgagcg aaaaagtgga tgcgctgacc aaaattatta ccggcatgaa ctatgatcag 2040

tttgataaaa aacgcagcgt gattgaagat accggccgcg aaaacgcgga acgcgaaaaa 2100

tttaaaaaaa ttattagcct gtatctgacc gtgatttatc atattctgaa aaacattgtg 2160

aacattaacg cgcgctatgt gattggcttt cattgcgtgg aacgcgatgc gcagctgtat 2220

aaagaaaaag gctatgatat taacctgaaa aaactggaag aaaaaggctt tagcagcgtg 2280

accaaactgt gcgcgggcat tgatgaaacc gcgccggata aacgcaaaga tgtggaaaaa 2340

gaaatggcgg aacgcgcgaa agaaagcatt gatagcctgg aaagcgcgaa cccgaaactg 2400

tatgcgaact atattaaata tagcgatgaa aaaaaagcgg aagaatttac ccgccagatt 2460

aaccgcgaaa aagcgaaaac cgcgctgaac gcgtatctgc gcaacaccaa atggaacgtg 2520

attattcgcg aagatctgct gcgcattgat aacaaaacct gcaccctgtt tcgcaacaaa 2580

gcggtgcatc tggaagtggc gcgctatgtg catgcgtata ttaacgatat tgcggaagtg 2640

aacagctatt ttcagctgta tcattatatt atgcagcgca ttattatgaa cgaacgctat 2700

gaaaaaagca gcggcaaagt gagcgaatat tttgatgcgg tgaacgatga aaaaaaatat 2760

aacgatcgcc tgctgaaact gctgtgcgtg ccgtttggct attgcattcc gcgctttaaa 2820

aacctgagca ttgaagcgct gtttgatcgc aacgaagcgg cgaaatttga taaagaaaaa 2880

aaaaaagtga gcggcaacag ctga 2904

<210> 10

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgatcgaga agaagaagag cttcgccaag ggcatgggcg tgaagagcac cctggtgagc 60

ggcagcaagg tgtacatgac caccttcgcc gagggcagcg acgccaggct ggagaagatc 120

gtggagggcg acagcatcag gagcgtgaac gagggcgagg ccttcagcgc cgagatggcc 180

gacaagaacg ccggctacaa gatcggcaac gccaagttca gccaccccaa gggctacgcc 240

gtggtggcca acaaccccct gtacaccggc cccgtgcagc aggacatgct gggcctgaag 300

gagaccctgg agaagaggta cttcggcgag agcgccgacg gcaacgacaa catctgcatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat cagcggcctg gacaaggaca tcatcggctt cggcaagttc 480

agcaccgtgt acacctacga cgagttcaag gaccccgagc accacagggc cgccttcaac 540

aacaacgaca agctgatcaa cgccatcaag gcccagtacg acgagttcga caacttcctg 600

gacaacccca ggctgggcta cttcggccag gccttcttca gcaaggaggg caggaactac 660

atcatcaact acggcaacga gtgctacgac atcctggccc tgctgagcgg cctgaggcac 720

tgggtggtgc acaacaacga ggaggagagc aggatcagca ggacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatcagc accctgaact acctgtacga caggatcacc 840

aacgagctga ccaacagctt cagcaagaac agcgccgcca acgtgaacta catcgccgag 900

accctgggca tcaaccccgc cgagttcgcc gagcagtact tcaggttcag catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgaggg aggtgatgct ggacaggaag 1020

gacatgagcg agatcaggaa gaaccacaag gtgttcgaca gcatcaggac caaggtgtac 1080

accatgatgg acttcgtgat ctacaggtac tacatcgagg aggacgccaa ggtggccgcc 1140

gccaacaaga gcctgcccga caacgagaag agcctgagcg agaaggacat cttcgtgatc 1200

aacctgaggg gcagcttcaa cgacgaccag aaggacgccc tgtactacga cgaggccaac 1260

aggatctgga ggaagctgga gaacatcatg cacaacatca aggagttcag gggcaacaag 1320

accagggagt acaagaagaa ggacgccccc aggctgccca ggatcctgcc cgccggcagg 1380

gacgtgagcg ccttcagcaa gctgatgtac gccctgacca tgttcctgga cggcaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagttcgaca acatccagag cttcctgaag 1500

gtgatgcccc tgatcggcgt gaacgccaag ttcgtggagg agtacgcctt cttcaaggac 1560

agcgccaaga tcgccgacga gctgaggctg atcaagagct tcgccaggat gggcgagccc 1620

atcgccgacg ccaggagggc catgtacatc gacgccatca ggatcctggg caccaacctg 1680

agctacgacg agctgaaggc cctggccgac accttcagcc tggacgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgaggaac ttcatcatca acaacgtgat cagcaacaag 1800

aggttccact acctgatcag gtacggcgac cccgcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggcagg atcgccgaca tccagaagaa gcagggccag 1920

aacggcaaga accagatcga caggtactac gagacctgca tcggcaagga caagggcaag 1980

agcgtgagcg agaaggtgga cgccctgacc aagatcatca ccggcatgaa ctacgaccag 2040

ttcgacaaga agaggagcgt gatcgaggac accggcaggg agaacgccga gagggagaag 2100

ttcaagaaga tcatcagcct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ccaggtacgt gatcggcttc cactgcgtgg agagggacgc ccagctgtac 2220

aaggagaagg gctacgacat caacctgaag aagctggagg agaagggctt cagcagcgtg 2280

accaagctgt gcgccggcat cgacgagacc gcccccgaca agaggaagga cgtggagaag 2340

gagatggccg agagggccaa ggagagcatc gacagcctgg agagcgccaa ccccaagctg 2400

tacgccaact acatcaagta cagcgacgag aagaaggccg aggagttcac caggcagatc 2460

aacagggaga aggccaagac cgccctgaac gcctacctga ggaacaccaa gtggaacgtg 2520

atcatcaggg aggacctgct gaggatcgac aacaagacct gcaccctgtt caggaacaag 2580

gccgtgcacc tggaggtggc caggtacgtg cacgcctaca tcaacgacat cgccgaggtg 2640

aacagctact tccagctgta ccactacatc atgcagagga tcatcatgaa cgagaggtac 2700

gagaagagca gcggcaaggt gagcgagtac ttcgacgccg tgaacgacga gaagaagtac 2760

aacgacaggc tgctgaagct gctgtgcgtg cccttcggct actgcatccc caggttcaag 2820

aacctgagca tcgaggccct gttcgacagg aacgaggccg ccaagttcga caaggagaag 2880

aagaaggtga gcggcaacag ctga 2904

<210> 11

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgatcgaga agaagaagag cttcgccaag ggcatgggcg tgaagagcac cctggtgagc 60

ggcagcaagg tgtacatgac caccttcgcc gagggcagcg acgccaggct ggagaagatc 120

gtggagggcg acagcatcag gagcgtgaac gagggcgagg ccttcagcgc cgagatggcc 180

gacaagaacg ccggctacaa gatcggcaac gccaagttca gccaccctaa gggctacgcc 240

gtggtggcca acaaccctct gtacaccggc cctgtgcagc aggacatgct gggcctgaag 300

gagaccctgg agaagaggta cttcggcgag agcgccgacg gcaacgacaa catctgcatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat cagcggcctg gacaaggaca tcatcggctt cggcaagttc 480

agcaccgtgt acacctacga cgagttcaag gaccctgagc accacagggc cgccttcaac 540

aacaacgaca agctgatcaa cgccatcaag gcccagtacg acgagttcga caacttcctg 600

gacaacccta ggctgggcta cttcggccag gccttcttca gcaaggaggg caggaactac 660

atcatcaact acggcaacga gtgctacgac atcctggccc tgctgagcgg cctgaggcac 720

tgggtggtgc acaacaacga ggaggagagc aggatcagca ggacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatcagc accctgaact acctgtacga caggatcacc 840

aacgagctga ccaacagctt cagcaagaac agcgccgcca acgtgaacta catcgccgag 900

accctgggca tcaaccctgc cgagttcgcc gagcagtact tcaggttcag catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgaggg aggtgatgct ggacaggaag 1020

gacatgagcg agatcaggaa gaaccacaag gtgttcgaca gcatcaggac caaggtgtac 1080

accatgatgg acttcgtgat ctacaggtac tacatcgagg aggacgccaa ggtggccgcc 1140

gccaacaaga gcctgcctga caacgagaag agcctgagcg agaaggacat cttcgtgatc 1200

aacctgaggg gcagcttcaa cgacgaccag aaggacgccc tgtactacga cgaggccaac 1260

aggatctgga ggaagctgga gaacatcatg cacaacatca aggagttcag gggcaacaag 1320

accagggagt acaagaagaa ggacgcccct aggctgccta ggatcctgcc tgccggcagg 1380

gacgtgagcg ccttcagcaa gctgatgtac gccctgacca tgttcctgga cggcaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagttcgaca acatccagag cttcctgaag 1500

gtgatgcctc tgatcggcgt gaacgccaag ttcgtggagg agtacgcctt cttcaaggac 1560

agcgccaaga tcgccgacga gctgaggctg atcaagagct tcgccaggat gggcgagcct 1620

atcgccgacg ccaggagggc catgtacatc gacgccatca ggatcctggg caccaacctg 1680

agctacgacg agctgaaggc cctggccgac accttcagcc tggacgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgaggaac ttcatcatca acaacgtgat cagcaacaag 1800

aggttccact acctgatcag gtacggcgac cctgcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggcagg atcgccgaca tccagaagaa gcagggccag 1920

aacggcaaga accagatcga caggtactac gagacctgca tcggcaagga caagggcaag 1980

agcgtgagcg agaaggtgga cgccctgacc aagatcatca ccggcatgaa ctacgaccag 2040

ttcgacaaga agaggagcgt gatcgaggac accggcaggg agaacgccga gagggagaag 2100

ttcaagaaga tcatcagcct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ccaggtacgt gatcggcttc cactgcgtgg agagggacgc ccagctgtac 2220

aaggagaagg gctacgacat caacctgaag aagctggagg agaagggctt cagcagcgtg 2280

accaagctgt gcgccggcat cgacgagacc gcccctgaca agaggaagga cgtggagaag 2340

gagatggccg agagggccaa ggagagcatc gacagcctgg agagcgccaa ccctaagctg 2400

tacgccaact acatcaagta cagcgacgag aagaaggccg aggagttcac caggcagatc 2460

aacagggaga aggccaagac cgccctgaac gcctacctga ggaacaccaa gtggaacgtg 2520

atcatcaggg aggacctgct gaggatcgac aacaagacct gcaccctgtt caggaacaag 2580

gccgtgcacc tggaggtggc caggtacgtg cacgcctaca tcaacgacat cgccgaggtg 2640

aacagctact tccagctgta ccactacatc atgcagagga tcatcatgaa cgagaggtac 2700

gagaagagca gcggcaaggt gagcgagtac ttcgacgccg tgaacgacga gaagaagtac 2760

aacgacaggc tgctgaagct gctgtgcgtg cctttcggct actgcatccc taggttcaag 2820

aacctgagca tcgaggccct gttcgacagg aacgaggccg ccaagttcga caaggagaag 2880

aagaaggtga gcggcaacag ctga 2904

<210> 12

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgattgaaa agaagaagtc ttttgctaag ggaatgggag ttaagtctac tcttgtttct 60

ggatctaagg tttatatgac tacttttgct gaaggatctg atgctagact tgaaaagatt 120

gttgaaggag attctattag atctgttaat gaaggagaag ctttttctgc tgaaatggct 180

gataagaatg ctggatataa gattggaaat gctaagtttt ctcatccaaa gggatatgct 240

gttgttgcta ataatccact ttatactgga ccagttcaac aagatatgct tggacttaag 300

gaaactcttg aaaagagata ttttggagaa tctgctgatg gaaatgataa tatttgtatt 360

caagttattc ataatattct tgatattgaa aagattcttg ctgaatatat tactaatgct 420

gcttatgctg ttaataatat ttctggactt gataaggata ttattggatt tggaaagttt 480

tctactgttt atacttatga tgaatttaag gatccagaac atcatagagc tgcttttaat 540

aataatgata agcttattaa tgctattaag gctcaatatg atgaatttga taattttctt 600

gataatccaa gacttggata ttttggacaa gctttttttt ctaaggaagg aagaaattat 660

attattaatt atggaaatga atgttatgat attcttgctc ttctttctgg acttagacat 720

tgggttgttc ataataatga agaagaatct agaatttcta gaacttggct ttataatctt 780

gataagaatc ttgataatga atatatttct actcttaatt atctttatga tagaattact 840

aatgaactta ctaattcttt ttctaagaat tctgctgcta atgttaatta tattgctgaa 900

actcttggaa ttaatccagc tgaatttgct gaacaatatt ttagattttc tattatgaag 960

gaacaaaaga atcttggatt taatattact aagcttagag aagttatgct tgatagaaag 1020

gatatgtctg aaattagaaa gaatcataag gtttttgatt ctattagaac taaggtttat 1080

actatgatgg attttgttat ttatagatat tatattgaag aagatgctaa ggttgctgct 1140

gctaataagt ctcttccaga taatgaaaag tctctttctg aaaaggatat ttttgttatt 1200

aatcttagag gatcttttaa tgatgatcaa aaggatgctc tttattatga tgaagctaat 1260

agaatttgga gaaagcttga aaatattatg cataatatta aggaatttag aggaaataag 1320

actagagaat ataagaagaa ggatgctcca agacttccaa gaattcttcc agctggaaga 1380

gatgtttctg ctttttctaa gcttatgtat gctcttacta tgtttcttga tggaaaggaa 1440

attaatgatc ttcttactac tcttattaat aagtttgata atattcaatc ttttcttaag 1500

gttatgccac ttattggagt taatgctaag tttgttgaag aatatgcttt ttttaaggat 1560

tctgctaaga ttgctgatga acttagactt attaagtctt ttgctagaat gggagaacca 1620

attgctgatg ctagaagagc tatgtatatt gatgctatta gaattcttgg aactaatctt 1680

tcttatgatg aacttaaggc tcttgctgat actttttctc ttgatgaaaa tggaaataag 1740

cttaagaagg gaaagcatgg aatgagaaat tttattatta ataatgttat ttctaataag 1800

agatttcatt atcttattag atatggagat ccagctcatc ttcatgaaat tgctaagaat 1860

gaagctgttg ttaagtttgt tcttggaaga attgctgata ttcaaaagaa gcaaggacaa 1920

aatggaaaga atcaaattga tagatattat gaaacttgta ttggaaagga taagggaaag 1980

tctgtttctg aaaaggttga tgctcttact aagattatta ctggaatgaa ttatgatcaa 2040

tttgataaga agagatctgt tattgaagat actggaagag aaaatgctga aagagaaaag 2100

tttaagaaga ttatttctct ttatcttact gttatttatc atattcttaa gaatattgtt 2160

aatattaatg ctagatatgt tattggattt cattgtgttg aaagagatgc tcaactttat 2220

aaggaaaagg gatatgatat taatcttaag aagcttgaag aaaagggatt ttcttctgtt 2280

actaagcttt gtgctggaat tgatgaaact gctccagata agagaaagga tgttgaaaag 2340

gaaatggctg aaagagctaa ggaatctatt gattctcttg aatctgctaa tccaaagctt 2400

tatgctaatt atattaagta ttctgatgaa aagaaggctg aagaatttac tagacaaatt 2460

aatagagaaa aggctaagac tgctcttaat gcttatctta gaaatactaa gtggaatgtt 2520

attattagag aagatcttct tagaattgat aataagactt gtactctttt tagaaataag 2580

gctgttcatc ttgaagttgc tagatatgtt catgcttata ttaatgatat tgctgaagtt 2640

aattcttatt ttcaacttta tcattatatt atgcaaagaa ttattatgaa tgaaagatat 2700

gaaaagtctt ctggaaaggt ttctgaatat tttgatgctg ttaatgatga aaagaagtat 2760

aatgatagac ttcttaagct tctttgtgtt ccatttggat attgtattcc aagatttaag 2820

aatctttcta ttgaagctct ttttgataga aatgaagctg ctaagtttga taaggaaaag 2880

aagaaggttt ctggaaattc ttga 2904

<210> 13

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgattgaaa agaagaagtc ttttgctaag ggtatgggtg ttaagtctac tttggtttct 60

ggttctaagg tttacatgac tacttttgct gaaggttctg atgctagatt ggaaaagatt 120

gttgaaggtg attctattag atctgttaac gaaggtgaag ctttttctgc tgaaatggct 180

gataagaacg ctggttacaa gattggtaac gctaagtttt ctcatccaaa gggttacgct 240

gttgttgcta acaacccatt gtacactggt ccagttcaac aagatatgtt gggtttgaag 300

gaaactttgg aaaagagata ctttggtgaa tctgctgatg gtaacgataa catttgtatt 360

caagttattc ataacatttt ggatattgaa aagattttgg ctgaatacat tactaacgct 420

gcttacgctg ttaacaacat ttctggtttg gataaggata ttattggttt tggtaagttt 480

tctactgttt acacttacga tgaatttaag gatccagaac atcatagagc tgcttttaac 540

aacaacgata agttgattaa cgctattaag gctcaatacg atgaatttga taactttttg 600

gataacccaa gattgggtta ctttggtcaa gctttttttt ctaaggaagg tagaaactac 660

attattaact acggtaacga atgttacgat attttggctt tgttgtctgg tttgagacat 720

tgggttgttc ataacaacga agaagaatct agaatttcta gaacttggtt gtacaacttg 780

gataagaact tggataacga atacatttct actttgaact acttgtacga tagaattact 840

aacgaattga ctaactcttt ttctaagaac tctgctgcta acgttaacta cattgctgaa 900

actttgggta ttaacccagc tgaatttgct gaacaatact ttagattttc tattatgaag 960

gaacaaaaga acttgggttt taacattact aagttgagag aagttatgtt ggatagaaag 1020

gatatgtctg aaattagaaa gaaccataag gtttttgatt ctattagaac taaggtttac 1080

actatgatgg attttgttat ttacagatac tacattgaag aagatgctaa ggttgctgct 1140

gctaacaagt ctttgccaga taacgaaaag tctttgtctg aaaaggatat ttttgttatt 1200

aacttgagag gttcttttaa cgatgatcaa aaggatgctt tgtactacga tgaagctaac 1260

agaatttgga gaaagttgga aaacattatg cataacatta aggaatttag aggtaacaag 1320

actagagaat acaagaagaa ggatgctcca agattgccaa gaattttgcc agctggtaga 1380

gatgtttctg ctttttctaa gttgatgtac gctttgacta tgtttttgga tggtaaggaa 1440

attaacgatt tgttgactac tttgattaac aagtttgata acattcaatc ttttttgaag 1500

gttatgccat tgattggtgt taacgctaag tttgttgaag aatacgcttt ttttaaggat 1560

tctgctaaga ttgctgatga attgagattg attaagtctt ttgctagaat gggtgaacca 1620

attgctgatg ctagaagagc tatgtacatt gatgctatta gaattttggg tactaacttg 1680

tcttacgatg aattgaaggc tttggctgat actttttctt tggatgaaaa cggtaacaag 1740

ttgaagaagg gtaagcatgg tatgagaaac tttattatta acaacgttat ttctaacaag 1800

agatttcatt acttgattag atacggtgat ccagctcatt tgcatgaaat tgctaagaac 1860

gaagctgttg ttaagtttgt tttgggtaga attgctgata ttcaaaagaa gcaaggtcaa 1920

aacggtaaga accaaattga tagatactac gaaacttgta ttggtaagga taagggtaag 1980

tctgtttctg aaaaggttga tgctttgact aagattatta ctggtatgaa ctacgatcaa 2040

tttgataaga agagatctgt tattgaagat actggtagag aaaacgctga aagagaaaag 2100

tttaagaaga ttatttcttt gtacttgact gttatttacc atattttgaa gaacattgtt 2160

aacattaacg ctagatacgt tattggtttt cattgtgttg aaagagatgc tcaattgtac 2220

aaggaaaagg gttacgatat taacttgaag aagttggaag aaaagggttt ttcttctgtt 2280

actaagttgt gtgctggtat tgatgaaact gctccagata agagaaagga tgttgaaaag 2340

gaaatggctg aaagagctaa ggaatctatt gattctttgg aatctgctaa cccaaagttg 2400

tacgctaact acattaagta ctctgatgaa aagaaggctg aagaatttac tagacaaatt 2460

aacagagaaa aggctaagac tgctttgaac gcttacttga gaaacactaa gtggaacgtt 2520

attattagag aagatttgtt gagaattgat aacaagactt gtactttgtt tagaaacaag 2580

gctgttcatt tggaagttgc tagatacgtt catgcttaca ttaacgatat tgctgaagtt 2640

aactcttact ttcaattgta ccattacatt atgcaaagaa ttattatgaa cgaaagatac 2700

gaaaagtctt ctggtaaggt ttctgaatac tttgatgctg ttaacgatga aaagaagtac 2760

aacgatagat tgttgaagtt gttgtgtgtt ccatttggtt actgtattcc aagatttaag 2820

aacttgtcta ttgaagcttt gtttgataga aacgaagctg ctaagtttga taaggaaaag 2880

aagaaggttt ctggtaactc ttga 2904

<210> 14

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgatcgaga agaagaagag cttcgccaag ggcatgggcg tgaagagcac cctggtgagc 60

ggcagcaagg tgtacatgac caccttcgcc gagggcagcg acgccaggct ggagaagatc 120

gtggagggcg acagcatcag gagcgtgaac gagggcgagg ccttcagcgc cgagatggcc 180

gacaagaacg ccggctacaa gatcggcaac gccaagttca gccaccccaa gggctacgcc 240

gtggtggcca acaaccccct gtacaccggc cccgtgcagc aggacatgct gggcctgaag 300

gagaccctgg agaagaggta cttcggcgag agcgccgacg gcaacgacaa catctgcatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat cagcggcctg gacaaggaca tcatcggctt cggcaagttc 480

agcaccgtgt acacctacga cgagttcaag gaccccgagc accacagggc cgccttcaac 540

aacaacgaca agctgatcaa cgccatcaag gcccagtacg acgagttcga caacttcctg 600

gacaacccca ggctgggcta cttcggccag gccttcttca gcaaggaggg caggaactac 660

atcatcaact acggcaacga gtgctacgac atcctggccc tgctgagcgg cctgaggcac 720

tgggtggtgc acaacaacga ggaggagagc aggatcagca ggacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatcagc accctgaact acctgtacga caggatcacc 840

aacgagctga ccaacagctt cagcaagaac agcgccgcca acgtgaacta catcgccgag 900

accctgggca tcaaccccgc cgagttcgcc gagcagtact tcaggttcag catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgaggg aggtgatgct ggacaggaag 1020

gacatgagcg agatcaggaa gaaccacaag gtgttcgaca gcatcaggac caaggtgtac 1080

accatgatgg acttcgtgat ctacaggtac tacatcgagg aggacgccaa ggtggccgcc 1140

gccaacaaga gcctgcccga caacgagaag agcctgagcg agaaggacat cttcgtgatc 1200

aacctgaggg gcagcttcaa cgacgaccag aaggacgccc tgtactacga cgaggccaac 1260

aggatctgga ggaagctgga gaacatcatg cacaacatca aggagttcag gggcaacaag 1320

accagggagt acaagaagaa ggacgccccc aggctgccca ggatcctgcc cgccggcagg 1380

gacgtgagcg ccttcagcaa gctgatgtac gccctgacca tgttcctgga cggcaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagttcgaca acatccagag cttcctgaag 1500

gtgatgcccc tgatcggcgt gaacgccaag ttcgtggagg agtacgcctt cttcaaggac 1560

agcgccaaga tcgccgacga gctgaggctg atcaagagct tcgccaggat gggcgagccc 1620

atcgccgacg ccaggagggc catgtacatc gacgccatca ggatcctggg caccaacctg 1680

agctacgacg agctgaaggc cctggccgac accttcagcc tggacgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgaggaac ttcatcatca acaacgtgat cagcaacaag 1800

aggttccact acctgatcag gtacggcgac cccgcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggcagg atcgccgaca tccagaagaa gcagggccag 1920

aacggcaaga accagatcga caggtactac gagacctgca tcggcaagga caagggcaag 1980

agcgtgagcg agaaggtgga cgccctgacc aagatcatca ccggcatgaa ctacgaccag 2040

ttcgacaaga agaggagcgt gatcgaggac accggcaggg agaacgccga gagggagaag 2100

ttcaagaaga tcatcagcct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ccaggtacgt gatcggcttc cactgcgtgg agagggacgc ccagctgtac 2220

aaggagaagg gctacgacat caacctgaag aagctggagg agaagggctt cagcagcgtg 2280

accaagctgt gcgccggcat cgacgagacc gcccccgaca agaggaagga cgtggagaag 2340

gagatggccg agagggccaa ggagagcatc gacagcctgg agagcgccaa ccccaagctg 2400

tacgccaact acatcaagta cagcgacgag aagaaggccg aggagttcac caggcagatc 2460

aacagggaga aggccaagac cgccctgaac gcctacctga ggaacaccaa gtggaacgtg 2520

atcatcaggg aggacctgct gaggatcgac aacaagacct gcaccctgtt caggaacaag 2580

gccgtgcacc tggaggtggc caggtacgtg cacgcctaca tcaacgacat cgccgaggtg 2640

aacagctact tccagctgta ccactacatc atgcagagga tcatcatgaa cgagaggtac 2700

gagaagagca gcggcaaggt gagcgagtac ttcgacgccg tgaacgacga gaagaagtac 2760

aacgacaggc tgctgaagct gctgtgcgtg cccttcggct actgcatccc caggttcaag 2820

aacctgagca tcgaggccct gttcgacagg aacgaggccg ccaagttcga caaggagaag 2880

aagaaggtga gcggcaacag ctga 2904

<210> 15

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgattgaaa aaaaaaaatc ttttgctaaa ggtatgggtg ttaaatctac tttggtttct 60

ggttctaaag tttatatgac tacttttgct gaaggttctg atgctagatt ggaaaaaatt 120

gttgaaggtg attctattag atctgttaat gaaggtgaag ctttttctgc tgaaatggct 180

gataaaaatg ctggttataa aattggtaat gctaaatttt ctcatccaaa aggttatgct 240

gttgttgcta ataatccatt gtatactggt ccagttcaac aagatatgtt gggtttgaaa 300

gaaactttgg aaaaaagata ttttggtgaa tctgctgatg gtaatgataa tatttgtatt 360

caagttattc ataatatttt ggatattgaa aaaattttgg ctgaatatat tactaatgct 420

gcttatgctg ttaataatat ttctggtttg gataaagata ttattggttt tggtaaattt 480

tctactgttt atacttatga tgaatttaaa gatccagaac atcatagagc tgcttttaat 540

aataatgata aattgattaa tgctattaaa gctcaatatg atgaatttga taattttttg 600

gataatccaa gattgggtta ttttggtcaa gctttttttt ctaaagaagg tagaaattat 660

attattaatt atggtaatga atgttatgat attttggctt tgttgtctgg tttgagacat 720

tgggttgttc ataataatga agaagaatct agaatttcta gaacttggtt gtataatttg 780

gataaaaatt tggataatga atatatttct actttgaatt atttgtatga tagaattact 840

aatgaattga ctaattcttt ttctaaaaat tctgctgcta atgttaatta tattgctgaa 900

actttgggta ttaatccagc tgaatttgct gaacaatatt ttagattttc tattatgaaa 960

gaacaaaaaa atttgggttt taatattact aaattgagag aagttatgtt ggatagaaaa 1020

gatatgtctg aaattagaaa aaatcataaa gtttttgatt ctattagaac taaagtttat 1080

actatgatgg attttgttat ttatagatat tatattgaag aagatgctaa agttgctgct 1140

gctaataaat ctttgccaga taatgaaaaa tctttgtctg aaaaagatat ttttgttatt 1200

aatttgagag gttcttttaa tgatgatcaa aaagatgctt tgtattatga tgaagctaat 1260

agaatttgga gaaaattgga aaatattatg cataatatta aagaatttag aggtaataaa 1320

actagagaat ataaaaaaaa agatgctcca agattgccaa gaattttgcc agctggtaga 1380

gatgtttctg ctttttctaa attgatgtat gctttgacta tgtttttgga tggtaaagaa 1440

attaatgatt tgttgactac tttgattaat aaatttgata atattcaatc ttttttgaaa 1500

gttatgccat tgattggtgt taatgctaaa tttgttgaag aatatgcttt ttttaaagat 1560

tctgctaaaa ttgctgatga attgagattg attaaatctt ttgctagaat gggtgaacca 1620

attgctgatg ctagaagagc tatgtatatt gatgctatta gaattttggg tactaatttg 1680

tcttatgatg aattgaaagc tttggctgat actttttctt tggatgaaaa tggtaataaa 1740

ttgaaaaaag gtaaacatgg tatgagaaat tttattatta ataatgttat ttctaataaa 1800

agatttcatt atttgattag atatggtgat ccagctcatt tgcatgaaat tgctaaaaat 1860

gaagctgttg ttaaatttgt tttgggtaga attgctgata ttcaaaaaaa acaaggtcaa 1920

aatggtaaaa atcaaattga tagatattat gaaacttgta ttggtaaaga taaaggtaaa 1980

tctgtttctg aaaaagttga tgctttgact aaaattatta ctggtatgaa ttatgatcaa 2040

tttgataaaa aaagatctgt tattgaagat actggtagag aaaatgctga aagagaaaaa 2100

tttaaaaaaa ttatttcttt gtatttgact gttatttatc atattttgaa aaatattgtt 2160

aatattaatg ctagatatgt tattggtttt cattgtgttg aaagagatgc tcaattgtat 2220

aaagaaaaag gttatgatat taatttgaaa aaattggaag aaaaaggttt ttcttctgtt 2280

actaaattgt gtgctggtat tgatgaaact gctccagata aaagaaaaga tgttgaaaaa 2340

gaaatggctg aaagagctaa agaatctatt gattctttgg aatctgctaa tccaaaattg 2400

tatgctaatt atattaaata ttctgatgaa aaaaaagctg aagaatttac tagacaaatt 2460

aatagagaaa aagctaaaac tgctttgaat gcttatttga gaaatactaa atggaatgtt 2520

attattagag aagatttgtt gagaattgat aataaaactt gtactttgtt tagaaataaa 2580

gctgttcatt tggaagttgc tagatatgtt catgcttata ttaatgatat tgctgaagtt 2640

aattcttatt ttcaattgta tcattatatt atgcaaagaa ttattatgaa tgaaagatat 2700

gaaaaatctt ctggtaaagt ttctgaatat tttgatgctg ttaatgatga aaaaaaatat 2760

aatgatagat tgttgaaatt gttgtgtgtt ccatttggtt attgtattcc aagatttaaa 2820

aatttgtcta ttgaagcttt gtttgataga aatgaagctg ctaaatttga taaagaaaaa 2880

aaaaaagttt ctggtaattc ttga 2904

<210> 16

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgattgaaa aaaaaaaatc ttttgctaaa ggtatgggtg ttaaatctac tttagtttct 60

ggttctaaag tttatatgac tacttttgct gaaggttctg atgctcgttt agaaaaaatt 120

gttgaaggtg attctattcg ttctgttaat gaaggtgaag ctttttctgc tgaaatggct 180

gataaaaatg ctggttataa aattggtaat gctaaatttt ctcatcctaa aggttatgct 240

gttgttgcta ataatccttt atatactggt cctgttcaac aagatatgtt aggtttaaaa 300

gaaactttag aaaaacgtta ttttggtgaa tctgctgatg gtaatgataa tatttgtatt 360

caagttattc ataatatttt agatattgaa aaaattttag ctgaatatat tactaatgct 420

gcttatgctg ttaataatat ttctggttta gataaagata ttattggttt tggtaaattt 480

tctactgttt atacttatga tgaatttaaa gatcctgaac atcatcgtgc tgcttttaat 540

aataatgata aattaattaa tgctattaaa gctcaatatg atgaatttga taatttttta 600

gataatcctc gtttaggtta ttttggtcaa gctttttttt ctaaagaagg tcgtaattat 660

attattaatt atggtaatga atgttatgat attttagctt tattatctgg tttacgtcat 720

tgggttgttc ataataatga agaagaatct cgtatttctc gtacttggtt atataattta 780

gataaaaatt tagataatga atatatttct actttaaatt atttatatga tcgtattact 840

aatgaattaa ctaattcttt ttctaaaaat tctgctgcta atgttaatta tattgctgaa 900

actttaggta ttaatcctgc tgaatttgct gaacaatatt ttcgtttttc tattatgaaa 960

gaacaaaaaa atttaggttt taatattact aaattacgtg aagttatgtt agatcgtaaa 1020

gatatgtctg aaattcgtaa aaatcataaa gtttttgatt ctattcgtac taaagtttat 1080

actatgatgg attttgttat ttatcgttat tatattgaag aagatgctaa agttgctgct 1140

gctaataaat ctttacctga taatgaaaaa tctttatctg aaaaagatat ttttgttatt 1200

aatttacgtg gttcttttaa tgatgatcaa aaagatgctt tatattatga tgaagctaat 1260

cgtatttggc gtaaattaga aaatattatg cataatatta aagaatttcg tggtaataaa 1320

actcgtgaat ataaaaaaaa agatgctcct cgtttacctc gtattttacc tgctggtcgt 1380

gatgtttctg ctttttctaa attaatgtat gctttaacta tgtttttaga tggtaaagaa 1440

attaatgatt tattaactac tttaattaat aaatttgata atattcaatc ttttttaaaa 1500

gttatgcctt taattggtgt taatgctaaa tttgttgaag aatatgcttt ttttaaagat 1560

tctgctaaaa ttgctgatga attacgttta attaaatctt ttgctcgtat gggtgaacct 1620

attgctgatg ctcgtcgtgc tatgtatatt gatgctattc gtattttagg tactaattta 1680

tcttatgatg aattaaaagc tttagctgat actttttctt tagatgaaaa tggtaataaa 1740

ttaaaaaaag gtaaacatgg tatgcgtaat tttattatta ataatgttat ttctaataaa 1800

cgttttcatt atttaattcg ttatggtgat cctgctcatt tacatgaaat tgctaaaaat 1860

gaagctgttg ttaaatttgt tttaggtcgt attgctgata ttcaaaaaaa acaaggtcaa 1920

aatggtaaaa atcaaattga tcgttattat gaaacttgta ttggtaaaga taaaggtaaa 1980

tctgtttctg aaaaagttga tgctttaact aaaattatta ctggtatgaa ttatgatcaa 2040

tttgataaaa aacgttctgt tattgaagat actggtcgtg aaaatgctga acgtgaaaaa 2100

tttaaaaaaa ttatttcttt atatttaact gttatttatc atattttaaa aaatattgtt 2160

aatattaatg ctcgttatgt tattggtttt cattgtgttg aacgtgatgc tcaattatat 2220

aaagaaaaag gttatgatat taatttaaaa aaattagaag aaaaaggttt ttcttctgtt 2280

actaaattat gtgctggtat tgatgaaact gctcctgata aacgtaaaga tgttgaaaaa 2340

gaaatggctg aacgtgctaa agaatctatt gattctttag aatctgctaa tcctaaatta 2400

tatgctaatt atattaaata ttctgatgaa aaaaaagctg aagaatttac tcgtcaaatt 2460

aatcgtgaaa aagctaaaac tgctttaaat gcttatttac gtaatactaa atggaatgtt 2520

attattcgtg aagatttatt acgtattgat aataaaactt gtactttatt tcgtaataaa 2580

gctgttcatt tagaagttgc tcgttatgtt catgcttata ttaatgatat tgctgaagtt 2640

aattcttatt ttcaattata tcattatatt atgcaacgta ttattatgaa tgaacgttat 2700

gaaaaatctt ctggtaaagt ttctgaatat tttgatgctg ttaatgatga aaaaaaatat 2760

aatgatcgtt tattaaaatt attatgtgtt ccttttggtt attgtattcc tcgttttaaa 2820

aatttatcta ttgaagcttt atttgatcgt aatgaagctg ctaaatttga taaagaaaaa 2880

aaaaaagttt ctggtaattc ttga 2904

<210> 17

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgatcgaga agaagaagtc cttcgctaag ggtatgggtg tgaagtccac cctggtgtcc 60

ggttccaagg tgtacatgac caccttcgct gagggttccg acgctcgtct ggagaagatc 120

gtggagggtg actccatccg ttccgtgaac gagggtgagg ctttctccgc tgagatggct 180

gacaagaacg ctggttacaa gatcggtaac gctaagttct cccaccctaa gggttacgct 240

gtggtggcta acaaccctct gtacaccggt cctgtgcagc aggacatgct gggtctgaag 300

gagaccctgg agaagcgtta cttcggtgag tccgctgacg gtaacgacaa catctgcatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ctgagtacat caccaacgct 420

gcttacgctg tgaacaacat ctccggtctg gacaaggaca tcatcggttt cggtaagttc 480

tccaccgtgt acacctacga cgagttcaag gaccctgagc accaccgtgc tgctttcaac 540

aacaacgaca agctgatcaa cgctatcaag gctcagtacg acgagttcga caacttcctg 600

gacaaccctc gtctgggtta cttcggtcag gctttcttct ccaaggaggg tcgtaactac 660

atcatcaact acggtaacga gtgctacgac atcctggctc tgctgtccgg tctgcgtcac 720

tgggtggtgc acaacaacga ggaggagtcc cgtatctccc gtacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatctcc accctgaact acctgtacga ccgtatcacc 840

aacgagctga ccaactcctt ctccaagaac tccgctgcta acgtgaacta catcgctgag 900

accctgggta tcaaccctgc tgagttcgct gagcagtact tccgtttctc catcatgaag 960

gagcagaaga acctgggttt caacatcacc aagctgcgtg aggtgatgct ggaccgtaag 1020

gacatgtccg agatccgtaa gaaccacaag gtgttcgact ccatccgtac caaggtgtac 1080

accatgatgg acttcgtgat ctaccgttac tacatcgagg aggacgctaa ggtggctgct 1140

gctaacaagt ccctgcctga caacgagaag tccctgtccg agaaggacat cttcgtgatc 1200

aacctgcgtg gttccttcaa cgacgaccag aaggacgctc tgtactacga cgaggctaac 1260

cgtatctggc gtaagctgga gaacatcatg cacaacatca aggagttccg tggtaacaag 1320

acccgtgagt acaagaagaa ggacgctcct cgtctgcctc gtatcctgcc tgctggtcgt 1380

gacgtgtccg ctttctccaa gctgatgtac gctctgacca tgttcctgga cggtaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagttcgaca acatccagtc cttcctgaag 1500

gtgatgcctc tgatcggtgt gaacgctaag ttcgtggagg agtacgcttt cttcaaggac 1560

tccgctaaga tcgctgacga gctgcgtctg atcaagtcct tcgctcgtat gggtgagcct 1620

atcgctgacg ctcgtcgtgc tatgtacatc gacgctatcc gtatcctggg taccaacctg 1680

tcctacgacg agctgaaggc tctggctgac accttctccc tggacgagaa cggtaacaag 1740

ctgaagaagg gtaagcacgg tatgcgtaac ttcatcatca acaacgtgat ctccaacaag 1800

cgtttccact acctgatccg ttacggtgac cctgctcacc tgcacgagat cgctaagaac 1860

gaggctgtgg tgaagttcgt gctgggtcgt atcgctgaca tccagaagaa gcagggtcag 1920

aacggtaaga accagatcga ccgttactac gagacctgca tcggtaagga caagggtaag 1980

tccgtgtccg agaaggtgga cgctctgacc aagatcatca ccggtatgaa ctacgaccag 2040

ttcgacaaga agcgttccgt gatcgaggac accggtcgtg agaacgctga gcgtgagaag 2100

ttcaagaaga tcatctccct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ctcgttacgt gatcggtttc cactgcgtgg agcgtgacgc tcagctgtac 2220

aaggagaagg gttacgacat caacctgaag aagctggagg agaagggttt ctcctccgtg 2280

accaagctgt gcgctggtat cgacgagacc gctcctgaca agcgtaagga cgtggagaag 2340

gagatggctg agcgtgctaa ggagtccatc gactccctgg agtccgctaa ccctaagctg 2400

tacgctaact acatcaagta ctccgacgag aagaaggctg aggagttcac ccgtcagatc 2460

aaccgtgaga aggctaagac cgctctgaac gcttacctgc gtaacaccaa gtggaacgtg 2520

atcatccgtg aggacctgct gcgtatcgac aacaagacct gcaccctgtt ccgtaacaag 2580

gctgtgcacc tggaggtggc tcgttacgtg cacgcttaca tcaacgacat cgctgaggtg 2640

aactcctact tccagctgta ccactacatc atgcagcgta tcatcatgaa cgagcgttac 2700

gagaagtcct ccggtaaggt gtccgagtac ttcgacgctg tgaacgacga gaagaagtac 2760

aacgaccgtc tgctgaagct gctgtgcgtg cctttcggtt actgcatccc tcgtttcaag 2820

aacctgtcca tcgaggctct gttcgaccgt aacgaggctg ctaagttcga caaggagaag 2880

aagaaggtgt ccggtaactc ctga 2904

<210> 18

<211> 2904

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgatcgaga agaagaagtc cttcgccaag ggcatgggcg tgaagtccac cctggtgtcc 60

ggctccaagg tgtacatgac caccttcgcc gagggctccg acgccaggct ggagaagatc 120

gtggagggcg actccatcag gtccgtgaac gagggcgagg ccttctccgc cgagatggcc 180

gacaagaacg ccggctacaa gatcggcaac gccaagttct cccacccgaa gggctacgcc 240

gtggtggcca acaacccgct gtacaccggc ccggtgcagc aggacatgct gggcctgaag 300

gagaccctgg agaagaggta cttcggcgag tccgccgacg gcaacgacaa catctgcatc 360

caggtgatcc acaacatcct ggacatcgag aagatcctgg ccgagtacat caccaacgcc 420

gcctacgccg tgaacaacat ctccggcctg gacaaggaca tcatcggctt cggcaagttc 480

tccaccgtgt acacctacga cgagttcaag gacccggagc accacagggc cgccttcaac 540

aacaacgaca agctgatcaa cgccatcaag gcccagtacg acgagttcga caacttcctg 600

gacaacccga ggctgggcta cttcggccag gccttcttct ccaaggaggg caggaactac 660

atcatcaact acggcaacga gtgctacgac atcctggccc tgctgtccgg cctgaggcac 720

tgggtggtgc acaacaacga ggaggagtcc aggatctcca ggacctggct gtacaacctg 780

gacaagaacc tggacaacga gtacatctcc accctgaact acctgtacga caggatcacc 840

aacgagctga ccaactcctt ctccaagaac tccgccgcca acgtgaacta catcgccgag 900

accctgggca tcaacccggc cgagttcgcc gagcagtact tcaggttctc catcatgaag 960

gagcagaaga acctgggctt caacatcacc aagctgaggg aggtgatgct ggacaggaag 1020

gacatgtccg agatcaggaa gaaccacaag gtgttcgact ccatcaggac caaggtgtac 1080

accatgatgg acttcgtgat ctacaggtac tacatcgagg aggacgccaa ggtggccgcc 1140

gccaacaagt ccctgccgga caacgagaag tccctgtccg agaaggacat cttcgtgatc 1200

aacctgaggg gctccttcaa cgacgaccag aaggacgccc tgtactacga cgaggccaac 1260

aggatctgga ggaagctgga gaacatcatg cacaacatca aggagttcag gggcaacaag 1320

accagggagt acaagaagaa ggacgccccg aggctgccga ggatcctgcc ggccggcagg 1380

gacgtgtccg ccttctccaa gctgatgtac gccctgacca tgttcctgga cggcaaggag 1440

atcaacgacc tgctgaccac cctgatcaac aagttcgaca acatccagtc cttcctgaag 1500

gtgatgccgc tgatcggcgt gaacgccaag ttcgtggagg agtacgcctt cttcaaggac 1560

tccgccaaga tcgccgacga gctgaggctg atcaagtcct tcgccaggat gggcgagccg 1620

atcgccgacg ccaggagggc catgtacatc gacgccatca ggatcctggg caccaacctg 1680

tcctacgacg agctgaaggc cctggccgac accttctccc tggacgagaa cggcaacaag 1740

ctgaagaagg gcaagcacgg catgaggaac ttcatcatca acaacgtgat ctccaacaag 1800

aggttccact acctgatcag gtacggcgac ccggcccacc tgcacgagat cgccaagaac 1860

gaggccgtgg tgaagttcgt gctgggcagg atcgccgaca tccagaagaa gcagggccag 1920

aacggcaaga accagatcga caggtactac gagacctgca tcggcaagga caagggcaag 1980

tccgtgtccg agaaggtgga cgccctgacc aagatcatca ccggcatgaa ctacgaccag 2040

ttcgacaaga agaggtccgt gatcgaggac accggcaggg agaacgccga gagggagaag 2100

ttcaagaaga tcatctccct gtacctgacc gtgatctacc acatcctgaa gaacatcgtg 2160

aacatcaacg ccaggtacgt gatcggcttc cactgcgtgg agagggacgc ccagctgtac 2220

aaggagaagg gctacgacat caacctgaag aagctggagg agaagggctt ctcctccgtg 2280

accaagctgt gcgccggcat cgacgagacc gccccggaca agaggaagga cgtggagaag 2340

gagatggccg agagggccaa ggagtccatc gactccctgg agtccgccaa cccgaagctg 2400

tacgccaact acatcaagta ctccgacgag aagaaggccg aggagttcac caggcagatc 2460

aacagggaga aggccaagac cgccctgaac gcctacctga ggaacaccaa gtggaacgtg 2520

atcatcaggg aggacctgct gaggatcgac aacaagacct gcaccctgtt caggaacaag 2580

gccgtgcacc tggaggtggc caggtacgtg cacgcctaca tcaacgacat cgccgaggtg 2640

aactcctact tccagctgta ccactacatc atgcagagga tcatcatgaa cgagaggtac 2700

gagaagtcct ccggcaaggt gtccgagtac ttcgacgccg tgaacgacga gaagaagtac 2760

aacgacaggc tgctgaagct gctgtgcgtg ccgttcggct actgcatccc gaggttcaag 2820

aacctgtcca tcgaggccct gttcgacagg aacgaggccg ccaagttcga caaggagaag 2880

aagaaggtgt ccggcaactc ctga 2904

<210> 19

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtgggtgccg tggctgtcac acttgctcgc 30

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gtctgtggaa gcgtgtccca tcctcctccg 30

<210> 21

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caatcgagac cctggtggac atcttccagg 30

<210> 22

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggagtgtgtg cccactgagg agtccaacat 30

<210> 23

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

acctcagttc cccttcaaag gggtggatga 30

<210> 24

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

taggacctgc cagtgctcag gcaacttcat 30

<210> 25

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gattagctga ctcttccgaa taaactcca 29

<210> 26

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ggagtagagg caggcgtcgt cccggaagtt 30

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gtgcccttcc cttgacagtt gagcacacgc 30

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggctgtgcag gctggtgccc ttggccacac 30

<210> 29

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cagcatcccg gccgctgacc acacctgcca 30

<210> 30

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gtcccatcct cctccggcac gctgttgaag 30

<210> 31

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gtgatggtga ccctgccctc aatctcccga 30

<210> 32

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gcaccctgga tgctggtatc taagagatac 30

<210> 33

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gctgtcttgg gtgcattgga gccttgcctt 30

<210> 34

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ctccaccatg ccaagtggtc ccaggctgc 29

<210> 35

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ggcagaagga ggagggcaga atcatcacga 30

<210> 36

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gtgaagttca tggatgtcta tcagcgcagc tac 33

<210> 37

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

cctgatgaga tcgagtacat cttcaagcca 30

<210> 38

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

cccctgatgc gatgcggggg ctgctgcaat 30

<210> 39

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gaccaaagaa agatagagca agacaaga 28

<210> 40

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gtttgtacaa gatccgcaga cgtgtaaatg t 31

<210> 41

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

agtttccaga tctctgatgg ccattttcct 30

<210> 42

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ggcaaaagaa tgctgcccac catggatg 28

<210> 43

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

tgtctccgaa aagaataagt tcttttctta cc 32

<210> 44

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tcagtctatg tcatccccac aggcacctat 30

<210> 45

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tggagagatg cagaaaactg tgacatttg 29

<210> 46

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ccatcttcag cagatgtgga attttgtctg a 31

<210> 47

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

agcagatcct tctcaaagta gcatgcacaa 30

<210> 48

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

atggtgattt cttcataaca tccaaggatc t 31

Claims

1. A CasRx preparation for silencing a gene of interest, comprising a CasRx mRNA and a sgRNA;

the sgRNA is set according to a target gene;

the mass ratio of CasRx mRNA to sgRNA is 5-100: 1.

2. The CasRx formulation of claim 1, wherein the CasRx mRNA further comprises a nucleotide sequence encoding a nuclear localization signal peptide.

3. A CasRx formulation according to claim 2, wherein said nucleotide sequence encoding a nuclear localization signal peptide is represented by SEQ ID No. 2.

4. A CasRx preparation according to claim 1, wherein the CasRx mRNA is from any one of SEQ ID Nos. 3 to 18.

5. The CasRx formulation according to claim 1, wherein when the gene of interest is the Pcsk 9gene, the sgRNA has a nucleotide sequence as shown in SEQ ID No.19 and/or SEQ ID No. 20.

6. The CasRx preparation according to claim 1, wherein when the gene of interest is the Vegfa gene, the nucleotide sequence of the sgRNA is shown in SEQ ID No.21 and/or SEQ ID No. 22.

7. A CasRx preparation according to claim 1, wherein when the gene of interest is the TYR gene, the nucleotide sequence of the sgRNA is as shown in SEQ ID No.23 and/or SEQ ID No. 24.

8. Use of a CasRx formulation as claimed in any one of claims 1 to 7 in gene silencing and/or RNA interference.