CN114634915A - Mutation library for constructing AAV serotype random insertion, kit and method thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a mutation library for constructing AAV serotype random insertion, a kit and a method thereof. The mutation library was constructed by random insertion of AAV serotypes. The kit comprises: a Cap cloning vector, a transposon containing a screening gene, a transposase, a specific endonuclease for removing the screening gene and a vector containing an AAV Rep coding gene sequence and ITR coding gene sequences at two ends. The invention solves the problems of low acquisition efficiency and low targeting property of new AAV serotypes in the prior art, establishes a random insertional mutation method which is relatively simple in operation, high in success rate and short in time period, and has important significance.

Description

Mutation library for constructing AAV serotype random insertion, kit and method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a kit and a method for constructing an AAV serotype random insertion mutation library.

Background

AAV is a DNA parvovirus, and is composed of a single-stranded DNA genome of 4.7kb in length. The naturally occurring AAV viral genome contains open reading frames of a capsid protein rep gene and a replication-associated protein Cap gene between ITRs at both ends. The modified AAV expression vector has Rep and Cap gene sequence eliminated to insert other target gene expression frame, and is co-transfected with packaging plasmid encoding Rep/Cap gene to transfect HEK-293T containing E1 gene, so as to package and release virus particle with invasion capacity including DNA and capsid.

Since AAV viruses are parvoviruses, the expression vector of recombinant adeno-associated virus (rAAV), which can be an effective foreign DNA delivery vector, is completely deleted between ITRs at both ends, in addition to ITR sequences that direct the replication of the genome and the assembly of the viral vector, and replaced with foreign transgene (transgene) sequences. In addition to reducing immunogenicity and cytotoxicity associated with transgene delivery in vivo, deletion of the gene encoding the viral protein has the greatest advantage of maximizing the capacity of the recombinant AAV to carry the transgene.

The establishment of AAV serotype mutation libraries is an important tool for screening serotypes with specific targeting, high transduction levels, or anti-neutralizing antibodies. The construction of Cap libraries in AAV serotype screening is difficult and technically complex. At present, the AAV serotype screening method mainly comprises the steps of carrying out random point mutation on a specific serotype, or inserting short peptides into fixed sites of the specific serotype, or carrying out shuffling by taking selected several serotype Cap genes as templates, or predicting the amino acid sequence of ancAAV by a reverse genetic screening method after carrying out sequence analysis on a plurality of serotypes, and selecting one of the ancAAV as a template to synthesize an ancAAV library by technologies such as random mutation and the like.

Although studies have shown that it is possible to obtain serotype mutants by constructing AAV mutant libraries using the prior art techniques and methods, there are problems such as complexity of sequence analysis, difficulty in constructing libraries, and a low proportion of mutants available. That is, these screening methods for obtaining random mutation libraries have extremely low probability of effective mutation.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a mutant library for constructing random insertion of AAV serotypes, a kit and a method thereof, which are used for solving the problem of low AAV acquisition efficiency of different serotypes in the prior art. Aiming at the difficulty in the prior art, the invention establishes a random insertion mutation method which is relatively simple in operation, high in success rate and short in time period, and has important significance.

In one aspect, the invention provides a library of mutations constructed by random insertion of AAV serotypes.

In another aspect, the present invention provides a method for constructing a library of AAV serotypes randomly inserted into mutations, the method comprising the steps of:

(1) under the action of transposase, randomly inserting transposons containing screening genes into a Cap cloning vector to obtain a mutation reaction product mixture;

(2) amplifying and screening the mutation reaction product inserted with the transposon to obtain a mixed cloning plasmid;

(3) extracting the mixed clone plasmid obtained in the step (2);

(4) carrying out enzyme digestion on the mixed cloning plasmid extracted in the step (3), recovering a Cap coding gene fragment and a blank cloning vector fragment which are introduced with mutation due to the insertion of transposon, carrying out ligation reaction, and obtaining a Cap cloning vector mixture only with Cap coding gene mutation, and marking as Cap insertion mutation mixed plasmid;

(5) removing the screening gene from the Cap inserted mutation mixed plasmid to obtain a mutated Cap cloning vector library;

(6) performing enzyme digestion on a mutated Cap cloning vector library, recovering a mutated Cap coding gene sequence, and inserting the mutated Cap coding gene sequence into a vector containing an AAV Rep coding gene sequence and ITR coding gene sequences at two ends to obtain a target plasmid insertion mutation library;

(7) inserting the target plasmid into the mutant library and transfecting cells together with the auxiliary vector to obtain the AAV mutant virus library.

In the step (1), the transposon, also called a "jumping gene", is a DNA sequence that can be independently copied or broken from the original position, circularized, inserted into another site, and used for regulating the subsequent gene. Transposase is an enzyme that performs a transposition function, and is generally encoded by a transposon, recognizes specific sequences at both ends of the transposon, can detach the transposon from adjacent sequences, and then inserts it into a new DNA target site without homology requirements.

The transposon of the present invention is selected from a type I transposon or a type II transposon.

Transposons are generally used in pairs with their corresponding transposases. In one embodiment, the transposon is selected from a transposable phage. Transposable phages are phages that alternate between lytic cycle and lysogenic type. For example, Mu phage are linear double stranded DNA molecules that have both the properties of temperate phages and the properties of transposons. The DNA can be inserted into almost any site of a target, and then the target mutation is induced. Illustratively, the transposon is a MuA transposon and the transposase is a MuA transposase.

In one embodiment, the transposon is selected from the TnA family. For example, the transposon may be selected from the group consisting of a Tn5 transposon, a Tn7 transposon, a Tn10 transposon and the like. Correspondingly, the transposase can be selected from the group consisting of Tn5 transposase, Tn7 transposase, Tn10 transposase, and the like.

In the present invention, the selection gene in the transposon is a drug resistance gene. The drug resistance gene is selected from prokaryotic antibiotic genes. Such as gentamicin resistance gene, kanamycin resistance gene, ampicillin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, etc.

Culturing in culture medium containing medicine in the step (2), and screening. The drug is a drug corresponding to a drug resistance gene.

In the invention, the Cap cloning vector refers to a cloning vector carrying a Cap coding gene sequence. Can be obtained by inserting the sequence of the Cap-encoding gene into a cloning vector. For example, the Cap coding gene sequence can be inserted into the PMD19T plasmid. In addition to the PMD19T plasmid, cloning vectors can be selected from the PMD18T plasmid, pUC19 plasmid, pUC57 and the like.

When the selection gene is removed in the step (5), the gene can be excised with a specific restriction enzyme. The skilled person can excise the drug resistance gene using a specific endonuclease directed against the drug resistance gene, preferably a single specific restriction endonuclease.

In the invention, the vector containing the AAV Rep coding gene sequence and the ITR coding gene sequences at two ends is obtained by inserting the Rep coding gene sequence and the ITR coding gene sequences at two ends into the vector. The vector is selected from the group consisting of f1 ori, pUC ori and resistance selection gene.

In step (7) above, the helper vector is selected from the pHelper plasmid.

In certain embodiments of the invention, the method of constructing a library of AAV serotypes randomly inserted into a mutation is a method of constructing a library of targeted AAV serotypes mutations.

The invention also provides a kit for constructing the AAV serotype random insertion mutation library based on the method, which comprises the following steps: a Cap cloning vector, a transposon containing a screening gene, a transposase, a specific endonuclease for removing the screening gene and a vector containing an AAV Rep coding gene sequence and ITR coding gene sequences at two ends.

The skilled person can excise the drug resistance gene using a specific endonuclease directed against the drug resistance gene. Preferably with a monospecific restriction endonuclease.

Further, the kit may also contain a helper vector, such as a phelprer plasmid, for packaging.

The invention also provides an AAV serotype mutant library obtained by the method.

The invention also provides an AAV targeting the spleen, the AAV comprising any one or more of the following features: an AAV obtained by inserting a sequence represented by any one of SEQ ID Nos. 26 to 31 between bases 69 to 70, 1943 to 1944, 269 to 270, 586 to 587, 189 to 190, 1898 to 1899 or 441 to 442 of a wild type AAV cap capsid protein-encoding gene.

Preferably, the AAV is obtained by inserting a sequence shown in SEQ ID NO.26 between 69 th to 70 th bases of a wild type AAV cap capsid protein coding gene.

Preferably, the AAV is obtained by inserting a sequence shown as SEQ ID NO.27 between 1943-1944 th bases of a wild type AAV cap capsid protein coding gene.

Preferably, the AAV is obtained by inserting a sequence shown in SEQ ID NO.28 between 269-270 th base of a wild type AAV cap capsid protein encoding gene.

Preferably, the AAV is obtained by inserting a sequence shown as SEQ ID NO.29 between 586-587 th bases or 1898-1899 th bases of a wild type AAV cap capsid protein encoding gene.

Preferably, the AAV is obtained by inserting a sequence shown as SEQ ID NO.30 between 189 th to 190 th bases of a wild type AAV cap capsid protein coding gene.

Preferably, the AAV is obtained by inserting a sequence shown as SEQ ID NO.31 between 441 th to 442 th base of a wild type AAV cap capsid protein coding gene.

The invention also provides the use of transposons and/or transposases for the construction of random insertion mutation libraries of AAV serotypes.

As described above, the kit and the method for constructing the AAV serotype mutant library have the following beneficial effects:

the method can establish a high-abundance mutation library in a short time, and the library is quickly verified in an in vitro model to obtain valuable mutation sites for subsequent further AAV (adeno-associated virus) phenotype analysis and directional modification. The invention can insert a fixed number of amino acid short peptides into random sites of a specific serotype by an efficient random insertion method. The method can simply and efficiently carry out insertion mutation small-sized library construction, virus packaging and function verification so as to search sites which can be modified by each serotype, analyze cell receptor binding sites and amino acid sites related to cell or tissue targeting, and can simply and efficiently prepare AAV serotypes with high targeting and high transduction efficiency.

Drawings

FIG. 1: schematic diagram of PMD19T carrier.

FIG. 2 is a schematic diagram: structural schematic diagram of MuA transposon.

FIG. 3: schematic representation of the insertion of the mutated PMD19T-cap plasmid at position 1476.

FIG. 4: schematic representation of the vector ITR used.

FIG. 5: the structure of the target carrier is shown schematically.

FIG. 6: cap mutant profile of liver and spleen tissues

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art. Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Example 1

This example provides a mutation library constructed by random insertion of AAV serotypes, and an exemplary method for constructing random insertion mutations of AAV serotypes, which essentially comprises the steps of:

step (1): PMD19T-Cap insertional mutagenesis reaction

The insertional mutagenesis reaction in this step is mainly to insert the MuA transposon into the Cap cloning vector (i.e. PMD19T-Cap plasmid DNA) under the action of MuA transposase, the central region of which encodes the drug resistance genes (ampicillin and gentamicin). The PMD19T-Cap plasmid is a PMD19T plasmid carrying Cap encoding gene sequences. The PMD19T plasmid is capable of clonal replication following transformation into a host cell and is therefore also referred to as a cloning vector. The sequence of the Cap coding gene is shown as SEQ ID NO.1, and specifically comprises the following steps:

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA。

the structure schematic diagram of the blank PMD19T plasmid is shown in FIG. 1, and specifically comprises the elements lacZ operator, AmpR resistance gene and ori replication origin.

The nucleotide sequence of the blank PMD19T plasmid is shown as SEQ ID NO.2, and specifically comprises the following steps:

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGATATCGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC。

the nucleotide sequence of the PMD19T plasmid carrying the Cap coding gene sequence, namely the PMD19T-Cap plasmid, is shown as SEQ ID NO.3, and specifically comprises the following steps:

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGATATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAAATCGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC。

a schematic diagram of the structure of the MuA transposon is shown in FIG. 2.

The nucleotide sequence of the MuA transposon is shown as SEQ ID NO.4

GATtacCTGGCGCGCCGCACGAAAAACGCGAAAGCGTTTCACGATAAATGCGAAAACGGATCGATCCTATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACttaggtggcggtacttgggtcgatatcaaagtgcatcacttcttcccgtatgcccaactttgtatagagagccactgcgggatcgtcaccgtaatctgcttgcacgtagatcacataagcaccaagcgcgttggcctcatgcttgaggagattgatgagcgcggtggcaatgccctgcctccggtgctcgccggagactgcgagatcatagatatagatctcactacgcggctgctcaaacctgggcagaacgtaagccgcgagagcgccaacaaccgcttcttggtcgaaggcagcaagcgcgatgaatgtcttactacggagcaagttcccgaggtaatcggagtccggctgatgttgggagtaggtggctacgtctccgaactcacgaccgaaaagatcaagagcagcccgcatggatttgacttggtcagggccgagcctacatgtgcgaatgatgcccatacttgagccacctaactttgttttagggcgactgccctgctgcgtaacatcgttgctgctgcgtaacatTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGTCGAAAAGGATCGATCCGTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGGCGCGCCAGgtaATC。

The nucleotide sequence of the MuA transposase is shown as SEQ ID NO.5

atggaactttgggtatcaccgaaagagtgtgcgaatcttcctggtttgccgaaaacatcggctggtgtgatttatgttgctaaaaagcaaggatggcaaaaccgcactagagcaggtgtcaaaggtggtaaagcaattgaatacaatgcgaactctttacctgttgaagcgaaagcggcgttattgctgagacaaggagagattgaaacaagcctggggtattttgaaatcgcccgccccacgctggaagcccatgattatgatcgtgaggcactgtggagcaaatgggataacgccagcgattcccagcgcagacttgctgaaaaatggttgcctgcggttcaggctgcagacgaaatgctgaaccaggggatttcaacgaaaacggcttttgcgaccgttgcagggcattaccaggtcagcgcatccactttgcgggacaagtattaccaggtacagaagtttgcgaagcctgactgggcggctgcacttgttgatggacgtggagcatcccgtcgcaatgttcacaaaagtgaatttgacgaggatgcctggcagtttctgattgcagattatctgcgaccggaaaaacccgctttccgcaaatgttatgagcgtctggaactggcagcccgcgagcatggctggagtattccctcccgtgccacggcctttcgccggattcagcaactggacgaggcaatggttgttgcctgtcgtgaaggtgaacatgcactgatgcatctgataccggcacagcagcgaactgtggaacacctggacgccatgcagtggatcaacggcgacggttatctgcataacgtctttgtacgctggtttaacggtgatgtgatccgtccgaaaacatggttctggcaggatgtgaaaacccgaaaaattctgggctggcgctgcgatgtgagcgagaacattgattcaattcgcctctcgttcatggatgttgtgactcgctacggtatcccggaggattttcacatcaccattgataacacccgtggtgctgcgaataaatggctgacgggaggcgcgcccaatcgctaccgctttaaggtaaaagaggacgatccaaaaggactgtttttactgatgggggcgaaaatgcactggacaagcgttgttgccggtaaaggctggggccaggcaaaacctgttgaacgtgctttcggtgttggtgggcttgaggaatacgttgataagcatccggcactggctggcgcatatacggggccaaatccgcaggcaaaacctgataactatggcgaccgcgctgttgatgcagagctgtttctgaaaacccttgccgaaggtgtggcgatgttcaatgccagaacaggccgtgaaacagaaatgtgcgggggcaaactctcgtttgatgatgttttcgagcgtgaatacgccagaacgattgtgcgtaagccaaccgaagaacaaaaacggatgctgttactgcctgccgaggcggtgaacgtttcacgcaaaggcgagtttacgcttaaagttggcggctcccttaaaggcgcgaaaaacgtttattacaacatggcattaatgaatgccggcgtgaaaaaagttgtggtcaggtttgatccgcagcagctacacagcacggtttattgctacaccctggacggtcggtttatctgtgaagcggaatgtctggcacctgttgcatttaatgatgctgcggcaggccgtgaatatcgccgccgccagaaacaactgaaatctgcgacgaaagcagccattaaggcgcagaaacaaatggacgcgctggaagttgctgaactgctgccgcagatagccgaaccagcagcaccagaatcacgaattgttggtattttccggccttccggtaatacggaacgggtgaagaatcaggagcgtgatgatgaatacgaaactgagcgtgatgaatatctgaatcattcgctggatattctggaacagaacagacgtaaaaaagccatttaa。

Mutation reaction 20. mu.l system included: PMD19T-Cap plasmid DNA 250ng, 1. mu.l MuA transposase, 1. mu.l MuA transposon, reaction buffer and ddH₂And O. The reaction conditions were 30 ℃ for 1h and 75 ℃ for 10 min.

Through the mutation reaction, the obtained mutation reaction product is a mixture and comprises PMD19T-Cap plasmid with only the part of Cap coding gene sequence mutated, PMD19T-Cap plasmid with only the part outside the Cap coding gene sequence mutated and PMD19T-Cap plasmid without any mutation. Among them, the schematic structure of PMD19T-Cap plasmid in which only the Cap-encoding gene sequence site was mutated is shown in FIG. 3.

Step (2): transformation of competent cells of E.coli Top10 with the product of the mutagenesis reaction

And (2) converting 10 mu l of the mutation reaction product obtained in the step (1) into 100 mu l of escherichia coli Top10 competent cells, converting by a calcium chloride method, adding 900 mu l of SOC (stress optical coefficient) non-resistant culture medium into the conversion product, carrying out shake culture at 37 ℃ and 220rpm for 1h, taking 20 mu l of a double-resistant plate coated with ampicillin and gentamicin, carrying out 37-degree overnight culture, and adding 4mL of ampicillin and gentamicin double-resistant SOC culture medium into the rest of the conversion solution to carry out overnight shake culture.

After this step, PMD19T-Cap plasmid without any mutation was removed, and mutant reaction products with MuA transposon inserted therein, including PMD19T-Cap plasmid in which only the part of Cap-encoding gene sequence was mutated, PMD19T-Cap plasmid in which only the part other than Cap-encoding gene sequence was mutated, collectively referred to as insertion mutation reaction products, can be selected.

The number of clones obtained from the insertional mutagenesis reaction product is shown in Table 1, and according to the random insertion principle, the total base number of PMD19T-Cap is 5k, and it is predicted that the insertional mutagenesis may cover each base site of Cap.

TABLE 1 number of clones obtained by insertion of the mutated reaction product

	20 μ l of the conversion product	1 conversion reaction	1 insertion mutation reaction
				Number of clones	1E+03	5E+04	1E+06

And (3): mixed clone plasmid extraction

And (3) taking the overnight culture product for amplification culture to about 20mL, culturing at 220rpm at 37 ℃ for 6h, and extracting plasmid DNA by using a Tiangen endotoxin-free plasmid extraction kit.

After this step, the cloned: PMD19T-Cap plasmid in which only the Cap-encoding gene sequence part is mutated, and PMD19T-Cap plasmid in which only the part other than the Cap-encoding gene sequence part is mutated, were extracted.

And (4): construction of Cap insertion mutation Mixed plasmid

And (3) enzyme digestion reaction: carrying out double enzyme digestion on EcoRI and HindIII of the mixed cloning plasmid extracted in the step (4), and cutting 3.4k of a mutant sequence containing an insert and 2.6k of a vector fragment which is not subjected to insertion mutation. The Cap sequence containing the insert refers to a Cap-encoding gene sequence mutated by the insertion of the MuA transposon. The non-insertionally mutated vector fragment corresponds to the original blank PMD19T plasmid.

And (3) glue recovery reaction: recovery of the mutated Cap-encoding gene sequence and the blank PMD19T plasmid was performed using the Gekko Swinhonis recovery kit.

And (3) connection reaction: 20 μ l ligation system included 100ng of blank PMD19T plasmid, the molar ratio of blank PMD19T plasmid to mutated Cap-encoding gene sequence was not 1: 2, ligase 2U, 10 Xligase Buffer 2. mu.l.

Transformation of the ligation products into Top10 competent cells and plasmid extraction were performed in the same manner as in steps (2) and (3), respectively.

Through the steps, the mutated Cap coding gene sequence is connected with a blank PMD19T plasmid, and a PMD19T plasmid carrying the mutated Cap coding gene sequence is obtained. Namely, a Cap cloning vector in which only a part of a Cap-encoding gene sequence is mutated. In this case, the sequence of the Cap-encoding gene to be mutated contains a drug resistance gene. Since the sequence mutations of the Cap-encoding gene may be different, it is also called Cap insertion mutation mixed plasmid. The number of clones of PMD19T-Cap mixed plasmid in which insertion mutation occurred only in the Cap region is shown in Table 2, and the total number of bases of Cap was 2.2k according to the random insertion principle, and it was predicted that insertion mutation could cover each base site of Cap.

TABLE 2 Cap insertion mutant PMD19T-Cap Mixed clone numbers

	20 μ l of the conversion product	1 conversion reaction
			Number of clones	50	2500

And (5): removal of inserted resistance genes

Enzyme digestion and recovery: and (3) carrying out enzyme digestion on the insertion mutation mixed plasmid extracted in the last step by using AscI endonuclease, carrying out gel electrophoresis separation on an enzyme digestion product, and recovering a target 5k fragment. The 5k fragment of interest here refers to the PMD19T plasmid with the mutated Cap-encoding gene sequence removed of the drug resistance genes (ampicillin and gentamicin). Simply referred to as "vector fragment".

Self-connection of the carrier: the 10. mu.l ligation system included 1. mu.l 10X T4 ligase Buffer, 1. mu.l ligase, and 8. mu.l vector fragment, ligation at 22 ℃ for 1h, and ligation at 16 ℃ for 1 h. The ligation product was a PMD19T plasmid with the entire mutated Cap-encoding gene sequence from which the drug resistance genes (ampicillin and gentamicin) were removed, designated as mutated _ Cap _ PMD19T plasmid.

Ligation products transformed Top10 competent cells: after the transformation, 900. mu.l of SOC non-resistant medium was added, shake cultivation was carried out at 37 ℃ and 220rpm for 1 hour, 20. mu.l of the ampicillin resistant plate was taken and subjected to 37 ℃ overnight cultivation, the gentamicin resistant plate was taken as a negative control, and the remaining transformation liquid was added to the ampicillin medium and subjected to shake cultivation overnight.

The plasmid extraction method is the same as the step (3).

10 clones were selected on overnight-cultured plates and subjected to one-generation sequencing with the PMD19T vector, i.e., cloned and replicated mutated _ Cap _ PMD19T plasmids M13F and M13R, respectively, and the sequencing results showed that the proportion of positive clones was 80%, and 8 Cap insertion sites were 771, 986, 1175, 1189, 1222, 1663, 2062 and 2096 bases, respectively. Correspondingly, the sequences of the 8 mutated Cap-encoding genes are:

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACTGGCGCGCCATCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.6)

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACTGGCGCGCCAGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.7)。

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTGGCGCGCCATTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.8)。

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTGGCGCGCCATGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.9)。

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACATGGCGCGCCAAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.10)。

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGAcattgTGGCGCGCCACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.11)。

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTgcagaTGGCGCGCCAGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.12)。

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAattcaTGGCGCGCCAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO.13)。

and (6): construction of a library of insertion mutations of the plasmid of interest

Enzyme digestion and recovery: the plasmid extracted in the previous step (i.e., mutated _ Cap _ PMD19T plasmid) was digested with SwaI and NotI enzymes, and the digested product was separated by gel electrophoresis to recover the desired 2.2k insert. The insert herein refers to a mutated Cap-encoding gene sequence cut from the mutated _ Cap _ PMD19T plasmid.

The objective plasmid is digested by SwaI and NotI enzymes, the digestion products are separated by gel electrophoresis, and the objective 4.9k vector fragment is recovered. The target plasmid is a vector containing AAV Rep-encoding gene sequences and ITR-encoding gene sequences at two ends. Namely, the target plasmid is obtained by inserting Rep coding gene sequences and ITR coding gene sequences at two ends into a JK-v101 vector, wherein the JK-v101 vector sequence is shown as SEQ ID NO.14, specifically

GCATGCgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacaCCTAGG。

The structural schematic diagram of the carrier containing AAV Rep coding gene sequence and ITR coding gene sequence at two ends is shown in figure 4.

The Rep coding gene sequence is shown as SEQ ID NO.15, and concretely comprises

ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA。

The two-end ITR coding gene sequence is shown as SEQ ID NO.16, specifically

GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT。

The nucleotide sequence of the target plasmid is shown as SEQ ID NO.17, and specifically comprises the following steps:

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggtctagagaggggtggagtcgtgacgtgaattacgtcatagggttagggaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggcccttttctttgtgcaatttgagaagggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaactgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgccgatggttatcttccagattggctcgaggacactctctctgaaggaataagacagtggtggaagctcaaacctggcccaccaccaccaaagcccgcagagcggcataaggacgacagcaggggtcttgtgcttcctgggtacaagtacctcggacccttcaacggactcgacaagggagagccggtcaacgaggcagacgccgcggccctcgagcacgacaaagcctacgaccggcagctcgacagcggagacaacccgtacctcaagtacaaccacgccgacgcggagtttcaggagcgccttaaagaagatacgtcttttgggggcaacctcggacgagcagtcttccaggcgaaaaagagggttcttgaacctctgggcctggttgaggaacctgttaagacggctccgggaaaaaagaggccggtagagcactctcctgtggagccagactcctcctcgggaaccggaaaggcgggccagcagcctgcaagaaaaagattgaattttggtcagactggagacgcagactcagtacctgacccccagcctctcggacagccaccagcagccccctctggtctgggaactaatacgatggctacaggcagtggcgcaccaatggcagacaataacgagggcgccgacggagtgggtaattcctcgggaaattggcattgcgattccacatggatgggcgacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaacaaccacctctacaaacaaatttccagccaatcaggagcctcgaacgacaatcactactttggctacagcaccccttgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcaaagactcatcaacaacaactggggattccgacccaagagactcaacttcaagctctttaacattcaagtcaaagaggtcacgcagaatgacggtacgacgacgattgccaataaccttaccagcacggttcaggtgtttactgactcggagtaccagctcccgtacgtcctcggctcggcgcatcaaggatgcctcccgccgttcccagcagacgtcttcatggtgccacagtatggatacctcaccctgaacaacgggagtcaggcagtaggacgctcttcattttactgcctggagtactttccttctcagatgctgcgtaccggaaacaactttaccttcagctacacttttgaggacgttcctttccacagcagctacgctcacagccagagtctggaccgtctcatgaatcctctcatcgaccagtacctgtattacttgagcagaacaaacactccaagtggaaccaccacgcagtcaaggcttcagttttctcaggccggagcgagtgacattcgggaccagtctaggaactggcttcctggaccctgttaccgccagcagcgagtatcaaagacatctgcggataacaacaacagtgaatactcgtggactggagctaccaagtaccacctcaatggcagagactctctggtgaatccgggcccggccatggcaagccacaaggacgatgaagaaaagttttttcctcagagcggggttctcatctttgggaagcaaggctcagagaaaacaaatgtggacattgaaaaggtcatgattacagacgaagaggaaatcaggacaaccaatcccgtggctacggagcagtatggttctgtatctaccaacctccagagaggcaacagacaagcagctaccgcagatgtcaacacacaaggcgttcttccaggcatggtctggcaggacagagatgtgtaccttcaggggcccatctgggcaaagattccacacacggacggacattttcacccctctcccctcatgggtggattcggacttaaacaccctcctccacagattctcatcaagaacaccccggtacctgcgaatccttcgaccaccttcagtgcggcaaagtttgcttccttcatcacacagtactccacgggacaggtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaaacgctggaatcccgaaattcagtacacttccaactacaacaagtctgttaatgtggactttactgtggacactaatggcgtgtattcagagcctcgccccattggcaccagatacctgactcgtaatctgtaattgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttatctagtttccatggctacgtagataagtagcatggcgggttaatcattaactacagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

and (3) connection reaction: the 10. mu.l ligation system included 1. mu.l 10X T4 ligase Buffer, 1. mu.l ligase, 100ng vector fragment (digested vector containing AAV Rep coding gene sequence and ITR coding gene sequences at both ends), 120ng insert (mutated Cap coding gene sequence cut from mutated _ Cap _ PMD19T plasmid) ligated at 22 ℃ for 1h, and 16 ℃ for 1 h.

Transformation of the ligation products into Top10 competent cells and plasmid extraction: the method is the same as the step (5).

Through the steps, the carrier containing AAV Rep coding gene sequence, ITR coding gene sequences at two ends and mutated Cap coding gene sequence is obtained. Simply referred to as "vector of interest" or "plasmid of interest inserted into a library of mutations". The structural schematic diagram of the "objective vector" or the "objective plasmid insertion mutation library" is shown in FIG. 5.

The number of mixed clones of the objective vector is shown in Table 3, and the total number of clones obtained is 7.5E +03, and it is predicted that the insertion mutation in the objective vector can cover all clones of the library of Cap insertion mutation PMD19T vector.

TABLE 3 number of mixed clones of the vectors of interest

	20 μ l of the conversion product	1 conversion reaction
			Number of clones	2.5E+02	7.5E+03

Example 2

And (2) carrying out vector containing AAV Rep coding gene sequence, ITR coding gene sequences at two ends and mutated Cap coding gene sequence obtained in the step (1). The target vector or the target plasmid is inserted into a mutation library for short, virus packaging and purification are carried out, the mutant AAV is obtained, and various performance parameters of the mutant AAV are investigated.

1. Mutant library virus packaging and purification

PEI transfection method two plasmid packaging systems: 1ml of Opti OMEM medium, 10. mu.g of the plasmid of interest obtained in step (6) of example 1 was inserted into the mutation library, 10. mu.g of pHelper plasmid, and after shaking and mixing, 60. mu.l of 1X PEI solution was added, and after shaking and mixing, the mixture was left to stand at room temperature for 20min to form a transfection mixture. The transfection mixture was added dropwise to 293T cells in a 10cm dish with 80% confluency at transfection and mixed in a cross. The carbon dioxide incubator was incubated at 37 ℃ overnight, and the medium was changed to 10ml of DMEM medium containing 10% FBS. And continuously culturing for 48 hours at 37 ℃ in a carbon dioxide incubator.

And (3) virus purification: recovering cell supernatant 72h after transfection, and centrifuging at 800g for 10min to remove cell precipitate; 1/4 volumes of 40% PEG8000 containing 2.5M NaCl was added to the supernatant and precipitated at 4 degrees for 2 h; centrifuging at 10000g 4 ℃ for 30min, and resuspending the precipitate with 500. mu.l PBS; adding 25U of totipotent nuclease, and digesting at 37 ℃ for 30 min; centrifuge at 3000g 4 ℃ for 15min and recover the supernatant. The purified virus was labeled P1 generation.

2. Mutant library virus titer detection

Virus genome titer by QPCR method: adding 1 mul of totipotent nuclease into 20 mul of purified virus, and incubating for 1h at 37 ℃; adding 1 mul proteinase K, and incubating at 60 ℃ for 20min to inactivate totipotent nuclease; incubating at 95 ℃ for 20min to inactivate the proteinase K; adding 90 mul PBS into 10 mul of treated sample, diluting by 10 times to be used as a sample to be detected for detection, and simultaneously carrying out QPCR amplification on the rep-containing plasmid DNA serving as a standard sample and the sample to be detected; the concentrations of the standard substances are respectively 1E +8, 1E +7, 1E +6, 1E +5, 1E +4 and 1E +3 copy/microliter. 20 μ l reaction System comprising ddH₂O, 1XTakara TB mix, 0.5. mu.l Rep-F and Rep-R primers (10. mu.M), 2. mu.l template DNA.

QPCR procedure: 95 ℃ for 1 min; 95 degrees 5S, 60 degrees 30S, 40 reaction cycles; fluorescence signal collection was performed at 60 degrees during the reaction, increasing the dissolution curve.

After the reaction is finished, the obtained sample copy number multiplied by 10000 is the virus genome copy number.

The titer and total amount of P1 generation virus obtained from packaging 110 cm dish were 1.23E +11vg/mL (genome copy number/mL) and 1.23E11 vg (genome copy number), respectively.

3. Mutant library virus passage and virus capsid sequence analysis

Virus passage:

293T cells were co-infected with 100. mu. l P1 passages of virus and adenovirus (MOI 0.5) with a cell confluence of 30% at infection and supernatants collected 72h after infection and purified according to virus purification protocol, labeled as passage P2.

The P2 generation virus and adenovirus were used to infect 293T cells in the same way, and the P3 generation virus was obtained in the same purification method.

4. Viral library capsid sequence analysis

PCR amplification is carried out by taking a sample treated by QPCR totipotenic nuclease of the P1 generation virus as a template, the sample is cloned to a PMD19T vector, primers M13F and M13R are used for sequencing, and the coverage repetition degree of different insertion sites of the AAV Cap which is successfully packaged is analyzed.

The insertion sites identified for the packaging virus are shown in table 4:

TABLE 4 random insertion site identification of packaging viruses

Insertion position	Insertion site region
		80aa thereafter	non-VR
86aa thereafter	non-VR
		592aa thereafter	VR-VIII
35aa thereafter	non-VR
		135aa thereafter	non-VR
492aa thereafter	VR-V

Second generation sequencing analysis abundance of each insertion site for the viruses of P1, P2 and P3 generations.

5. Verification of packaging viral transduction levels and Targeted changes

And inserting a targeting peptide segment into the identified site capable of carrying out insertion mutation, and verifying the targeting change after serotype modification.

In vivo targeting verification: injecting 150 mul mutant library virus into a mouse body by a tail vein injection mode, separating two tissues of a liver and a spleen after 3 weeks, extracting genome DNA of the 2 tissues, amplifying AAV Cap sequences in the 2 tissues by nested PCR, and arranging the obtained AAV Cap mixed sequences for high-throughput second-generation sequencing. Subsequently, the results of high-throughput next generation sequencing of the two tissues, liver and spleen, were analyzed by comparison.

Basic cap capsid sequences used for constructing mutant library viruses are mainly distributed in liver, skeletal muscle, eyes and nerve tissues before mutation, and comparative analysis of high-throughput second-generation sequencing results of the liver and the spleen tissues shows that after the basic cap capsid sequences are inserted into sequences of 15bp at some sites, the tissue infectivity of the basic cap capsid sequences is changed to spleen tissues from the liver, the skeletal muscle, the eyes and the nerve tissues. This phenomenon indicates that inserting a sequence at some sites is helpful for targeting, and also indicates that the mutation method is very helpful for subsequent screening of AAV cap capsid sequences with targeting.

The conditions and recovery mode for nested PCR amplification are as follows:

(1) the primer sequence is as follows:

F1:TGTCGTCAAAAAGGCGTATCAG(SEQ ID NO.18)

R1:CAACTGAAACGAATTAAACGG(SEQ ID NO.19)

F2:CGATCTGGTCAATGTGGATTTG(SEQ ID NO.20)

R2:GCAATTACAGATTACGAGTCAGG(SEQ ID NO.21)

(2) an amplification system:

(3) and (3) amplification procedure:

the procedure is as follows: storing at 94 ℃ for 3min, 94 ℃ for 30S, 55 ℃ for 30S, 72 ℃ for 2min, 30 cycles, 72 ℃ for 5min and 8 ℃.

(4) The amplification method comprises the following steps:

nested PCR: in the first round, F1 and R1 are used as primers, and a genome is used as a template for amplification; the second round of PCR was amplified using F1 and R1 as primers and the first round of PCR as a template.

(5) And (3) identifying the second round PCR solution obtained by adopting nested PCR amplification by adopting 1% agarose gel electrophoresis.

(6) And (3) directly carrying out PCR column recovery on the PCR liquid sample obtained by amplification by adopting a Tiangen DNA product purification kit, and arranging high-throughput second-generation sequencing on the recovered sample.

Results and analysis:

as can be seen from the liver and spleen tissue cap mutant profile (FIG. 6), some of the cap mutants only target the spleen but not the liver, and this result indicates that the targeting property can be indeed changed when a fixed sequence is inserted into a certain site of wild-type cap.

The sequences of cap mutants, which were more distributed in liver and spleen tissues in the distribution map, were individually analyzed in comparison and the results are shown in the following table:

it can be seen that TGCGGCCGCAGGTGG, TGCGGCCGCACTCAT, TGCGGCCGCACCGTA, TGCGGCCGCACCCCT, TGCGGCCGCAGAGAG, TGCGGCCGCACCCCT, TGCGGCCGCATAGAG are inserted at positions 69, 1943 (after 592 aa), 269 (after 86 aa), 586, 189, 1898 (after 592 aa), 441 (after 135 aa) of the wild cap gene sequence, respectively, which is more likely to infect spleen tissue.

Sequence listing

<110> Shanghai Jikai Gene medicine science and technology Co., Ltd

<120> a mutation library for constructing random insertion of AAV serotypes, and a kit and a method thereof

<150> 2020114821288

<151> 2020-12-15

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2208

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga aattcagtac 2100

acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160

tcagagcctc gccccattgg caccagatac ctgactcgta atctgtaa 2208

<210> 2

<211> 2692

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420

cctctagaga tatcgtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg 480

tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 540

aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 600

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 660

gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 720

cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 780

tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 840

aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 900

catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 960

caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 1020

ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 1080

aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 1140

gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 1200

cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 1260

ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 1320

tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 1380

tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 1440

cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 1500

tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 1560

tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 1620

tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 1680

cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 1740

ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1800

tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1860

gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1920

agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1980

atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 2040

tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 2100

gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 2160

agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 2220

cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 2280

ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 2340

ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 2400

actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 2460

ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 2520

atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 2580

caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 2640

attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc 2692

<210> 3

<211> 4900

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420

cctctagaga tatggctgcc gatggttatc ttccagattg gctcgaggac actctctctg 480

aaggaataag acagtggtgg aagctcaaac ctggcccacc accaccaaag cccgcagagc 540

ggcataagga cgacagcagg ggtcttgtgc ttcctgggta caagtacctc ggacccttca 600

acggactcga caagggagag ccggtcaacg aggcagacgc cgcggccctc gagcacgaca 660

aagcctacga ccggcagctc gacagcggag acaacccgta cctcaagtac aaccacgccg 720

acgcggagtt tcaggagcgc cttaaagaag atacgtcttt tgggggcaac ctcggacgag 780

cagtcttcca ggcgaaaaag agggttcttg aacctctggg cctggttgag gaacctgtta 840

agacggctcc gggaaaaaag aggccggtag agcactctcc tgtggagcca gactcctcct 900

cgggaaccgg aaaggcgggc cagcagcctg caagaaaaag attgaatttt ggtcagactg 960

gagacgcaga ctcagtacct gacccccagc ctctcggaca gccaccagca gccccctctg 1020

gtctgggaac taatacgatg gctacaggca gtggcgcacc aatggcagac aataacgagg 1080

gcgccgacgg agtgggtaat tcctcgggaa attggcattg cgattccaca tggatgggcg 1140

acagagtcat caccaccagc acccgaacct gggccctgcc cacctacaac aaccacctct 1200

acaaacaaat ttccagccaa tcaggagcct cgaacgacaa tcactacttt ggctacagca 1260

ccccttgggg gtattttgac ttcaacagat tccactgcca cttttcacca cgtgactggc 1320

aaagactcat caacaacaac tggggattcc gacccaagag actcaacttc aagctcttta 1380

acattcaagt caaagaggtc acgcagaatg acggtacgac gacgattgcc aataacctta 1440

ccagcacggt tcaggtgttt actgactcgg agtaccagct cccgtacgtc ctcggctcgg 1500

cgcatcaagg atgcctcccg ccgttcccag cagacgtctt catggtgcca cagtatggat 1560

acctcaccct gaacaacggg agtcaggcag taggacgctc ttcattttac tgcctggagt 1620

actttccttc tcagatgctg cgtaccggaa acaactttac cttcagctac acttttgagg 1680

acgttccttt ccacagcagc tacgctcaca gccagagtct ggaccgtctc atgaatcctc 1740

tcatcgacca gtacctgtat tacttgagca gaacaaacac tccaagtgga accaccacgc 1800

agtcaaggct tcagttttct caggccggag cgagtgacat tcgggaccag tctaggaact 1860

ggcttcctgg accctgttac cgccagcagc gagtatcaaa gacatctgcg gataacaaca 1920

acagtgaata ctcgtggact ggagctacca agtaccacct caatggcaga gactctctgg 1980

tgaatccggg cccggccatg gcaagccaca aggacgatga agaaaagttt tttcctcaga 2040

gcggggttct catctttggg aagcaaggct cagagaaaac aaatgtggac attgaaaagg 2100

tcatgattac agacgaagag gaaatcagga caaccaatcc cgtggctacg gagcagtatg 2160

gttctgtatc taccaacctc cagagaggca acagacaagc agctaccgca gatgtcaaca 2220

cacaaggcgt tcttccaggc atggtctggc aggacagaga tgtgtacctt caggggccca 2280

tctgggcaaa gattccacac acggacggac attttcaccc ctctcccctc atgggtggat 2340

tcggacttaa acaccctcct ccacagattc tcatcaagaa caccccggta cctgcgaatc 2400

cttcgaccac cttcagtgcg gcaaagtttg cttccttcat cacacagtac tccacgggac 2460

aggtcagcgt ggagatcgag tgggagctgc agaaggaaaa cagcaaacgc tggaatcccg 2520

aaattcagta cacttccaac tacaacaagt ctgttaatgt ggactttact gtggacacta 2580

atggcgtgta ttcagagcct cgccccattg gcaccagata cctgactcgt aatctgtaaa 2640

tcgtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga 2700

aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 2760

tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 2820

cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 2880

ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 2940

cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 3000

ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 3060

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 3120

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 3180

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 3240

gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 3300

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3360

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3420

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 3480

gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 3540

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 3600

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 3660

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 3720

tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 3780

aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 3840

taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 3900

gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 3960

agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 4020

cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 4080

tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 4140

gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 4200

agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 4260

gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 4320

atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 4380

gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 4440

tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 4500

atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 4560

agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 4620

gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 4680

cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 4740

tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 4800

ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 4860

ttaacctata aaaataggcg tatcacgagg ccctttcgtc 4900

<210> 4

<211> 1087

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gattacctgg cgcgccgcac gaaaaacgcg aaagcgtttc acgataaatg cgaaaacgga 60

tcgatcctat cgtcaattat tacctccacg gggagagcct gagcaaactg gcctcaggca 120

tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag 180

acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg 240

aatttctgcc attcatccgc ttattatcac ttaggtggcg gtacttgggt cgatatcaaa 300

gtgcatcact tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc 360

gtaatctgct tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag 420

attgatgagc gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata 480

gatatagatc tcactacgcg gctgctcaaa cctgggcaga acgtaagccg cgagagcgcc 540

aacaaccgct tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt 600

cccgaggtaa tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg 660

accgaaaaga tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt 720

gcgaatgatg cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt 780

aacatcgttg ctgctgcgta acattttagc ttccttagct cctgaaaatc tcgacaactc 840

aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg 900

ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac 960

accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggtcgaaaa 1020

ggatcgatcc gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtg cggcgcgcca 1080

ggtaatc 1087

<210> 5

<211> 1992

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaacttt gggtatcacc gaaagagtgt gcgaatcttc ctggtttgcc gaaaacatcg 60

gctggtgtga tttatgttgc taaaaagcaa ggatggcaaa accgcactag agcaggtgtc 120

aaaggtggta aagcaattga atacaatgcg aactctttac ctgttgaagc gaaagcggcg 180

ttattgctga gacaaggaga gattgaaaca agcctggggt attttgaaat cgcccgcccc 240

acgctggaag cccatgatta tgatcgtgag gcactgtgga gcaaatggga taacgccagc 300

gattcccagc gcagacttgc tgaaaaatgg ttgcctgcgg ttcaggctgc agacgaaatg 360

ctgaaccagg ggatttcaac gaaaacggct tttgcgaccg ttgcagggca ttaccaggtc 420

agcgcatcca ctttgcggga caagtattac caggtacaga agtttgcgaa gcctgactgg 480

gcggctgcac ttgttgatgg acgtggagca tcccgtcgca atgttcacaa aagtgaattt 540

gacgaggatg cctggcagtt tctgattgca gattatctgc gaccggaaaa acccgctttc 600

cgcaaatgtt atgagcgtct ggaactggca gcccgcgagc atggctggag tattccctcc 660

cgtgccacgg cctttcgccg gattcagcaa ctggacgagg caatggttgt tgcctgtcgt 720

gaaggtgaac atgcactgat gcatctgata ccggcacagc agcgaactgt ggaacacctg 780

gacgccatgc agtggatcaa cggcgacggt tatctgcata acgtctttgt acgctggttt 840

aacggtgatg tgatccgtcc gaaaacatgg ttctggcagg atgtgaaaac ccgaaaaatt 900

ctgggctggc gctgcgatgt gagcgagaac attgattcaa ttcgcctctc gttcatggat 960

gttgtgactc gctacggtat cccggaggat tttcacatca ccattgataa cacccgtggt 1020

gctgcgaata aatggctgac gggaggcgcg cccaatcgct accgctttaa ggtaaaagag 1080

gacgatccaa aaggactgtt tttactgatg ggggcgaaaa tgcactggac aagcgttgtt 1140

gccggtaaag gctggggcca ggcaaaacct gttgaacgtg ctttcggtgt tggtgggctt 1200

gaggaatacg ttgataagca tccggcactg gctggcgcat atacggggcc aaatccgcag 1260

gcaaaacctg ataactatgg cgaccgcgct gttgatgcag agctgtttct gaaaaccctt 1320

gccgaaggtg tggcgatgtt caatgccaga acaggccgtg aaacagaaat gtgcgggggc 1380

aaactctcgt ttgatgatgt tttcgagcgt gaatacgcca gaacgattgt gcgtaagcca 1440

accgaagaac aaaaacggat gctgttactg cctgccgagg cggtgaacgt ttcacgcaaa 1500

ggcgagttta cgcttaaagt tggcggctcc cttaaaggcg cgaaaaacgt ttattacaac 1560

atggcattaa tgaatgccgg cgtgaaaaaa gttgtggtca ggtttgatcc gcagcagcta 1620

cacagcacgg tttattgcta caccctggac ggtcggttta tctgtgaagc ggaatgtctg 1680

gcacctgttg catttaatga tgctgcggca ggccgtgaat atcgccgccg ccagaaacaa 1740

ctgaaatctg cgacgaaagc agccattaag gcgcagaaac aaatggacgc gctggaagtt 1800

gctgaactgc tgccgcagat agccgaacca gcagcaccag aatcacgaat tgttggtatt 1860

ttccggcctt ccggtaatac ggaacgggtg aagaatcagg agcgtgatga tgaatacgaa 1920

actgagcgtg atgaatatct gaatcattcg ctggatattc tggaacagaa cagacgtaaa 1980

aaagccattt aa 1992

<210> 6

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta ctggcgcgcc 780

atctacaaac aaatttccag ccaatcagga gcctcgaacg acaatcacta ctttggctac 840

agcacccctt gggggtattt tgacttcaac agattccact gccacttttc accacgtgac 900

tggcaaagac tcatcaacaa caactgggga ttccgaccca agagactcaa cttcaagctc 960

tttaacattc aagtcaaaga ggtcacgcag aatgacggta cgacgacgat tgccaataac 1020

cttaccagca cggttcaggt gtttactgac tcggagtacc agctcccgta cgtcctcggc 1080

tcggcgcatc aaggatgcct cccgccgttc ccagcagacg tcttcatggt gccacagtat 1140

ggatacctca ccctgaacaa cgggagtcag gcagtaggac gctcttcatt ttactgcctg 1200

gagtactttc cttctcagat gctgcgtacc ggaaacaact ttaccttcag ctacactttt 1260

gaggacgttc ctttccacag cagctacgct cacagccaga gtctggaccg tctcatgaat 1320

cctctcatcg accagtacct gtattacttg agcagaacaa acactccaag tggaaccacc 1380

acgcagtcaa ggcttcagtt ttctcaggcc ggagcgagtg acattcggga ccagtctagg 1440

aactggcttc ctggaccctg ttaccgccag cagcgagtat caaagacatc tgcggataac 1500

aacaacagtg aatactcgtg gactggagct accaagtacc acctcaatgg cagagactct 1560

ctggtgaatc cgggcccggc catggcaagc cacaaggacg atgaagaaaa gttttttcct 1620

cagagcgggg ttctcatctt tgggaagcaa ggctcagaga aaacaaatgt ggacattgaa 1680

aaggtcatga ttacagacga agaggaaatc aggacaacca atcccgtggc tacggagcag 1740

tatggttctg tatctaccaa cctccagaga ggcaacagac aagcagctac cgcagatgtc 1800

aacacacaag gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg 1860

cccatctggg caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt 1920

ggattcggac ttaaacaccc tcctccacag attctcatca agaacacccc ggtacctgcg 1980

aatccttcga ccaccttcag tgcggcaaag tttgcttcct tcatcacaca gtactccacg 2040

ggacaggtca gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 7

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtactggc gcgccaggta cgacgacgat tgccaataac 1020

cttaccagca cggttcaggt gtttactgac tcggagtacc agctcccgta cgtcctcggc 1080

tcggcgcatc aaggatgcct cccgccgttc ccagcagacg tcttcatggt gccacagtat 1140

ggatacctca ccctgaacaa cgggagtcag gcagtaggac gctcttcatt ttactgcctg 1200

gagtactttc cttctcagat gctgcgtacc ggaaacaact ttaccttcag ctacactttt 1260

gaggacgttc ctttccacag cagctacgct cacagccaga gtctggaccg tctcatgaat 1320

cctctcatcg accagtacct gtattacttg agcagaacaa acactccaag tggaaccacc 1380

acgcagtcaa ggcttcagtt ttctcaggcc ggagcgagtg acattcggga ccagtctagg 1440

aactggcttc ctggaccctg ttaccgccag cagcgagtat caaagacatc tgcggataac 1500

aacaacagtg aatactcgtg gactggagct accaagtacc acctcaatgg cagagactct 1560

ctggtgaatc cgggcccggc catggcaagc cacaaggacg atgaagaaaa gttttttcct 1620

cagagcgggg ttctcatctt tgggaagcaa ggctcagaga aaacaaatgt ggacattgaa 1680

aaggtcatga ttacagacga agaggaaatc aggacaacca atcccgtggc tacggagcag 1740

tatggttctg tatctaccaa cctccagaga ggcaacagac aagcagctac cgcagatgtc 1800

aacacacaag gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg 1860

cccatctggg caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt 1920

ggattcggac ttaaacaccc tcctccacag attctcatca agaacacccc ggtacctgcg 1980

aatccttcga ccaccttcag tgcggcaaag tttgcttcct tcatcacaca gtactccacg 2040

ggacaggtca gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 8

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattggcgc gccattcatt ttactgcctg 1200

gagtactttc cttctcagat gctgcgtacc ggaaacaact ttaccttcag ctacactttt 1260

gaggacgttc ctttccacag cagctacgct cacagccaga gtctggaccg tctcatgaat 1320

cctctcatcg accagtacct gtattacttg agcagaacaa acactccaag tggaaccacc 1380

acgcagtcaa ggcttcagtt ttctcaggcc ggagcgagtg acattcggga ccagtctagg 1440

aactggcttc ctggaccctg ttaccgccag cagcgagtat caaagacatc tgcggataac 1500

aacaacagtg aatactcgtg gactggagct accaagtacc acctcaatgg cagagactct 1560

ctggtgaatc cgggcccggc catggcaagc cacaaggacg atgaagaaaa gttttttcct 1620

cagagcgggg ttctcatctt tgggaagcaa ggctcagaga aaacaaatgt ggacattgaa 1680

aaggtcatga ttacagacga agaggaaatc aggacaacca atcccgtggc tacggagcag 1740

tatggttctg tatctaccaa cctccagaga ggcaacagac aagcagctac cgcagatgtc 1800

aacacacaag gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg 1860

cccatctggg caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt 1920

ggattcggac ttaaacaccc tcctccacag attctcatca agaacacccc ggtacctgcg 1980

aatccttcga ccaccttcag tgcggcaaag tttgcttcct tcatcacaca gtactccacg 2040

ggacaggtca gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 9

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagtg gcgcgccatg 1200

gagtactttc cttctcagat gctgcgtacc ggaaacaact ttaccttcag ctacactttt 1260

gaggacgttc ctttccacag cagctacgct cacagccaga gtctggaccg tctcatgaat 1320

cctctcatcg accagtacct gtattacttg agcagaacaa acactccaag tggaaccacc 1380

acgcagtcaa ggcttcagtt ttctcaggcc ggagcgagtg acattcggga ccagtctagg 1440

aactggcttc ctggaccctg ttaccgccag cagcgagtat caaagacatc tgcggataac 1500

aacaacagtg aatactcgtg gactggagct accaagtacc acctcaatgg cagagactct 1560

ctggtgaatc cgggcccggc catggcaagc cacaaggacg atgaagaaaa gttttttcct 1620

cagagcgggg ttctcatctt tgggaagcaa ggctcagaga aaacaaatgt ggacattgaa 1680

aaggtcatga ttacagacga agaggaaatc aggacaacca atcccgtggc tacggagcag 1740

tatggttctg tatctaccaa cctccagaga ggcaacagac aagcagctac cgcagatgtc 1800

aacacacaag gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg 1860

cccatctggg caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt 1920

ggattcggac ttaaacaccc tcctccacag attctcatca agaacacccc ggtacctgcg 1980

aatccttcga ccaccttcag tgcggcaaag tttgcttcct tcatcacaca gtactccacg 2040

ggacaggtca gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 10

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggaaa catggcgcgc caaaacaact ttaccttcag ctacactttt 1260

gaggacgttc ctttccacag cagctacgct cacagccaga gtctggaccg tctcatgaat 1320

cctctcatcg accagtacct gtattacttg agcagaacaa acactccaag tggaaccacc 1380

acgcagtcaa ggcttcagtt ttctcaggcc ggagcgagtg acattcggga ccagtctagg 1440

aactggcttc ctggaccctg ttaccgccag cagcgagtat caaagacatc tgcggataac 1500

aacaacagtg aatactcgtg gactggagct accaagtacc acctcaatgg cagagactct 1560

ctggtgaatc cgggcccggc catggcaagc cacaaggacg atgaagaaaa gttttttcct 1620

cagagcgggg ttctcatctt tgggaagcaa ggctcagaga aaacaaatgt ggacattgaa 1680

aaggtcatga ttacagacga agaggaaatc aggacaacca atcccgtggc tacggagcag 1740

tatggttctg tatctaccaa cctccagaga ggcaacagac aagcagctac cgcagatgtc 1800

aacacacaag gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg 1860

cccatctggg caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt 1920

ggattcggac ttaaacaccc tcctccacag attctcatca agaacacccc ggtacctgcg 1980

aatccttcga ccaccttcag tgcggcaaag tttgcttcct tcatcacaca gtactccacg 2040

ggacaggtca gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 11

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca aatgtggaca ttgtggcgcg ccacattgaa 1680

aaggtcatga ttacagacga agaggaaatc aggacaacca atcccgtggc tacggagcag 1740

tatggttctg tatctaccaa cctccagaga ggcaacagac aagcagctac cgcagatgtc 1800

aacacacaag gcgttcttcc aggcatggtc tggcaggaca gagatgtgta ccttcagggg 1860

cccatctggg caaagattcc acacacggac ggacattttc acccctctcc cctcatgggt 1920

ggattcggac ttaaacaccc tcctccacag attctcatca agaacacccc ggtacctgcg 1980

aatccttcga ccaccttcag tgcggcaaag tttgcttcct tcatcacaca gtactccacg 2040

ggacaggtca gcgtggagat cgagtgggag ctgcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 12

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gatggcgcgc cagcagaagg aaaacagcaa acgctggaat 2100

cccgaaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 13

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga aattcatggc 2100

gcgccaattc agtacacttc caactacaac aagtctgtta atgtggactt tactgtggac 2160

actaatggcg tgtattcaga gcctcgcccc attggcacca gatacctgac tcgtaatctg 2220

taa 2223

<210> 14

<211> 2572

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcatgcgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 60

ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 120

gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 180

ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 240

ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 300

acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 360

actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 420

atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 480

aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 540

tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 600

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 660

ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 720

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 780

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 840

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 900

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 960

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 1020

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 1080

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 1140

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 1200

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 1260

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1320

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1380

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1440

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1500

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1560

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1620

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1680

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1740

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1800

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1860

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1920

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1980

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 2040

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 2100

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 2160

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 2220

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 2280

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 2340

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2400

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2460

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2520

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacaccta gg 2572

<210> 15

<211> 1866

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180

cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240

caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 300

aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 420

gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840

cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800

ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860

caataa 1866

<210> 16

<211> 143

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 60

acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag agggagtggc 120

caactccatc actaggggtt cct 143

<210> 17

<211> 7285

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggtctag agaggggtgg agtcgtgacg tgaattacgt 180

catagggtta gggaggtcct gtattagagg tcacgtgagt gttttgcgac attttgcgac 240

accatgtggt cacgctgggt atttaagccc gagtgagcac gcagggtctc cattttgaag 300

cgggaggttt gaacgcgcag ccgccatgcc ggggttttac gagattgtga ttaaggtccc 360

cagcgacctt gacgagcatc tgcccggcat ttctgacagc tttgtgaact gggtggccga 420

gaaggaatgg gagttgccgc cagattctga catggatctg aatctgattg agcaggcacc 480

cctgaccgtg gccgagaagc tgcagcgcga ctttctgacg gaatggcgcc gtgtgagtaa 540

ggccccggag gcccttttct ttgtgcaatt tgagaaggga gagagctact tccacatgca 600

cgtgctcgtg gaaaccaccg gggtgaaatc catggttttg ggacgtttcc tgagtcagat 660

tcgcgaaaaa ctgattcaga gaatttaccg cgggatcgag ccgactttgc caaactggtt 720

cgcggtcaca aagaccagaa atggcgccgg aggcgggaac aaggtggtgg atgagtgcta 780

catccccaat tacttgctcc ccaaaaccca gcctgagctc cagtgggcgt ggactaatat 840

ggaacagtat ttaagcgcct gtttgaatct cacggagcgt aaacggttgg tggcgcagca 900

tctgacgcac gtgtcgcaga cgcaggagca gaacaaagag aatcagaatc ccaattctga 960

tgcgccggtg atcagatcaa aaacttcagc caggtacatg gagctggtcg ggtggctcgt 1020

ggacaagggg attacctcgg agaagcagtg gatccaggag gaccaggcct catacatctc 1080

cttcaatgcg gcctccaact cgcggtccca aatcaaggct gccttggaca atgcgggaaa 1140

gattatgagc ctgactaaaa ccgcccccga ctacctggtg ggccagcagc ccgtggagga 1200

catttccagc aatcggattt ataaaatttt ggaactaaac gggtacgatc cccaatatgc 1260

ggcttccgtc tttctgggat gggccacgaa aaagttcggc aagaggaaca ccatctggct 1320

gtttgggcct gcaactaccg ggaagaccaa catcgcggag gccatagccc acactgtgcc 1380

cttctacggg tgcgtaaact ggaccaatga gaactttccc ttcaacgact gtgtcgacaa 1440

gatggtgatc tggtgggagg aggggaagat gaccgccaag gtcgtggagt cggccaaagc 1500

cattctcgga ggaagcaagg tgcgcgtgga ccagaaatgc aagtcctcgg cccagataga 1560

cccgactccc gtgatcgtca cctccaacac caacatgtgc gccgtgattg acgggaactc 1620

aacgaccttc gaacaccagc agccgttgca agaccggatg ttcaaatttg aactcacccg 1680

ccgtctggat catgactttg ggaaggtcac caagcaggaa gtcaaagact ttttccggtg 1740

ggcaaaggat cacgtggttg aggtggagca tgaattctac gtcaaaaagg gtggagccaa 1800

gaaaagaccc gcccccagtg acgcagatat aagtgagccc aaacgggtgc gcgagtcagt 1860

tgcgcagcca tcgacgtcag acgcggaagc ttcgatcaac tacgcagaca ggtaccaaaa 1920

caaatgttct cgtcacgtgg gcatgaatct gatgctgttt ccctgcagac aatgcgagag 1980

aatgaatcag aattcaaata tctgcttcac tcacggacag aaagactgtt tagagtgctt 2040

tcccgtgtca gaatctcaac ccgtttctgt cgtcaaaaag gcgtatcaga aactgtgcta 2100

cattcatcat atcatgggaa aggtgccaga cgcttgcact gcctgcgatc tggtcaatgt 2160

ggatttggat gactgcatct ttgaacaata aatgatttaa atcaggtatg gctgccgatg 2220

gttatcttcc agattggctc gaggacactc tctctgaagg aataagacag tggtggaagc 2280

tcaaacctgg cccaccacca ccaaagcccg cagagcggca taaggacgac agcaggggtc 2340

ttgtgcttcc tgggtacaag tacctcggac ccttcaacgg actcgacaag ggagagccgg 2400

tcaacgaggc agacgccgcg gccctcgagc acgacaaagc ctacgaccgg cagctcgaca 2460

gcggagacaa cccgtacctc aagtacaacc acgccgacgc ggagtttcag gagcgcctta 2520

aagaagatac gtcttttggg ggcaacctcg gacgagcagt cttccaggcg aaaaagaggg 2580

ttcttgaacc tctgggcctg gttgaggaac ctgttaagac ggctccggga aaaaagaggc 2640

cggtagagca ctctcctgtg gagccagact cctcctcggg aaccggaaag gcgggccagc 2700

agcctgcaag aaaaagattg aattttggtc agactggaga cgcagactca gtacctgacc 2760

cccagcctct cggacagcca ccagcagccc cctctggtct gggaactaat acgatggcta 2820

caggcagtgg cgcaccaatg gcagacaata acgagggcgc cgacggagtg ggtaattcct 2880

cgggaaattg gcattgcgat tccacatgga tgggcgacag agtcatcacc accagcaccc 2940

gaacctgggc cctgcccacc tacaacaacc acctctacaa acaaatttcc agccaatcag 3000

gagcctcgaa cgacaatcac tactttggct acagcacccc ttgggggtat tttgacttca 3060

acagattcca ctgccacttt tcaccacgtg actggcaaag actcatcaac aacaactggg 3120

gattccgacc caagagactc aacttcaagc tctttaacat tcaagtcaaa gaggtcacgc 3180

agaatgacgg tacgacgacg attgccaata accttaccag cacggttcag gtgtttactg 3240

actcggagta ccagctcccg tacgtcctcg gctcggcgca tcaaggatgc ctcccgccgt 3300

tcccagcaga cgtcttcatg gtgccacagt atggatacct caccctgaac aacgggagtc 3360

aggcagtagg acgctcttca ttttactgcc tggagtactt tccttctcag atgctgcgta 3420

ccggaaacaa ctttaccttc agctacactt ttgaggacgt tcctttccac agcagctacg 3480

ctcacagcca gagtctggac cgtctcatga atcctctcat cgaccagtac ctgtattact 3540

tgagcagaac aaacactcca agtggaacca ccacgcagtc aaggcttcag ttttctcagg 3600

ccggagcgag tgacattcgg gaccagtcta ggaactggct tcctggaccc tgttaccgcc 3660

agcagcgagt atcaaagaca tctgcggata acaacaacag tgaatactcg tggactggag 3720

ctaccaagta ccacctcaat ggcagagact ctctggtgaa tccgggcccg gccatggcaa 3780

gccacaagga cgatgaagaa aagttttttc ctcagagcgg ggttctcatc tttgggaagc 3840

aaggctcaga gaaaacaaat gtggacattg aaaaggtcat gattacagac gaagaggaaa 3900

tcaggacaac caatcccgtg gctacggagc agtatggttc tgtatctacc aacctccaga 3960

gaggcaacag acaagcagct accgcagatg tcaacacaca aggcgttctt ccaggcatgg 4020

tctggcagga cagagatgtg taccttcagg ggcccatctg ggcaaagatt ccacacacgg 4080

acggacattt tcacccctct cccctcatgg gtggattcgg acttaaacac cctcctccac 4140

agattctcat caagaacacc ccggtacctg cgaatccttc gaccaccttc agtgcggcaa 4200

agtttgcttc cttcatcaca cagtactcca cgggacaggt cagcgtggag atcgagtggg 4260

agctgcagaa ggaaaacagc aaacgctgga atcccgaaat tcagtacact tccaactaca 4320

acaagtctgt taatgtggac tttactgtgg acactaatgg cgtgtattca gagcctcgcc 4380

ccattggcac cagatacctg actcgtaatc tgtaattgct tgttaatcaa taaaccgttt 4440

aattcgtttc agttgaactt tggtctctgc gtatttcttt cttatctagt ttccatggct 4500

acgtagataa gtagcatggc gggttaatca ttaactacag cggccgcagg aacccctagt 4560

gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa 4620

ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg 4680

cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 4740

catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 4800

gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 4860

ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 4920

ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg 4980

ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 5040

gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 5100

tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 5160

gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta 5220

tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 5280

ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 5340

gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 5400

gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 5460

gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 5520

tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 5580

caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 5640

ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 5700

gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 5760

aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 5820

ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 5880

atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 5940

gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 6000

gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 6060

atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 6120

aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 6180

actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 6240

aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 6300

tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 6360

ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 6420

agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 6480

tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 6540

aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 6600

gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 6660

atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 6720

gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 6780

gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 6840

tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 6900

accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 6960

ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 7020

cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 7080

agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 7140

ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 7200

tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 7260

ttttgctggc cttttgctca catgt 7285

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgtcgtcaaa aaggcgtatc ag 22

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

caactgaaac gaattaaacg g 21

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cgatctggtc aatgtggatt tg 22

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gcaattacag attacgagtc agg 23

<210> 22

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tgcggccgca cctgt 15

<210> 23

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tgcggccgca tccgg 15

<210> 24

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tgcggccgca accag 15

<210> 25

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tgcggccgca accac 15

<210> 26

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgcggccgca ggtgg 15

<210> 27

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tgcggccgca ctcat 15

<210> 28

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tgcggccgca ccgta 15

<210> 29

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tgcggccgca cccct 15

<210> 30

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tgcggccgca gagag 15

<210> 31

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tgcggccgca tagag 15

Claims (14)

1. A library of mutations constructed by random insertion of AAV serotypes.

2. A method of constructing a library of AAV serotypes randomly inserted into mutations, said method comprising the steps of:

(1) under the action of transposase, randomly inserting transposons containing screening genes into a Cap cloning vector to obtain a mutation reaction product mixture;

(2) amplifying and screening the mutation reaction product inserted with the transposon to obtain a mixed cloning plasmid;

(3) extracting the mixed clone plasmid obtained in the step (2);

(4) carrying out enzyme digestion on the mixed cloning plasmid extracted in the step (3), recovering a Cap coding gene fragment and a blank cloning vector fragment which are introduced with mutation due to the insertion of transposon, carrying out ligation reaction, and obtaining a Cap cloning vector mixture only with Cap coding gene mutation, and marking as Cap insertion mutation mixed plasmid;

(5) removing the screening gene from the Cap inserted mutation mixed plasmid to obtain a mutated Cap cloning vector library;

(6) carrying out enzyme digestion on the mutated Cap cloning vector library, recovering the mutated Cap coding gene sequence, and inserting the mutated Cap coding gene sequence into a vector containing an AAVRep coding gene sequence and ITR coding gene sequences at two ends to obtain a target plasmid insertion mutation library;

(7) inserting the target plasmid into the mutant library and transfecting cells together with the auxiliary vector to obtain the AAV mutant virus library.

3. The method of claim 2, wherein said transposon is selected from a transposable phage or the TnA family.

4. The method of claim 3, wherein the transposon is selected from the group consisting of a MuA transposon, a Tn5 transposon, a Tn7 transposon and a Tn10 transposon.

5. The method according to claim 2, wherein the selection gene in the transposon is a drug resistance gene; preferably, the selection gene in the transposon is a prokaryotic antibiotic gene.

6. The method according to claim 2, wherein the Cap cloning vector is a cloning vector carrying a Cap-encoding gene sequence, and is obtained by inserting the Cap-encoding gene sequence into the cloning vector.

7. The method of claim 6, wherein the cloning vector is selected from the group consisting of a PMD19T plasmid, a PMD18T plasmid, a pUC19 plasmid, and a pUC57 plasmid.

8. The method according to claim 2, wherein the removal of the screening gene in step (5) is performed by excising the screening gene with a specific restriction enzyme.

9. The method according to claim 2, wherein in step (6), the vector containing the AAV Rep-encoding gene sequences and ITR-encoding gene sequences at both ends is obtained by inserting the Rep-encoding gene sequences and ITR-encoding gene sequences at both ends into the vector.

10. A library of AAV serotype mutations obtainable by the method of any one of claims 2 to 9.

11. An AAV that targets the spleen, wherein said AAV comprises any one or more of the following characteristics: an AAV obtained by inserting a sequence represented by any one of SEQ ID nos. 26 to 31 between 69 th to 70 th bases, 1943 th to 1944 th bases, 269 th to 270 th bases, 586 th to 587 th bases, 189 th to 190 th bases, 1898 th to 1899 th bases and 441 th to 442 th bases of a wild type AAV cap capsid protein-encoding gene.

12. An AAV targeting the spleen according to claim 11, wherein said AAV is selected from any one or more of:

1) the AAV is obtained by inserting a sequence shown as SEQID NO.26 between 69 th to 70 th base groups of a wild AAV cap capsid protein coding gene;

2) the AAV is obtained by inserting a sequence shown as SEQ ID NO.27 between 1943-1944 th base of a wild AAV cap capsid protein coding gene;

3) the AAV is obtained by inserting a sequence shown as SEQ ID NO.28 between 269-270 th base groups of a wild AAV cap capsid protein coding gene;

4) the AAV is obtained by inserting a sequence shown as SEQ ID NO.29 between 586 th-587 th bases or 1898 th-1899 th bases of a wild AAV cap capsid protein coding gene;

5) the AAV is obtained by inserting a sequence shown as SEQ ID NO.30 between 189 th to 190 th base groups of a wild AAV cap capsid protein coding gene;

6) the AAV is obtained by inserting a sequence shown in SEQ ID NO.31 between 441 th to 442 th base groups of a wild AAV cap capsid protein coding gene.

13. A kit for constructing a library of AAV serotypes randomly inserted into mutations comprising: a Cap cloning vector, a transposon containing a screening gene, a transposase, a specific endonuclease for removing the screening gene and a vector containing an AAV Rep coding gene sequence and ITR coding gene sequences at two ends.

14. Use of a transposon and/or transposase in the construction of a library of AAV serotype mutations.

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