CN113667017A - Method capable of improving homologous recombination efficiency of CRISPR/Cas9 system and application - Google Patents

Abstract

The invention provides a method and application for improving homologous recombination efficiency of a CRISPR/Cas9 system, which comprises the following steps: cas9-TetR fusion proteins, guide RNAs, vectors containing tetracycline operator (tetO) elements, and a method of homologous recombination using the above system. The method does not need to carry out additional chemical modification and purification on the recombinant vector or the Cas9 protein, and only needs to construct the homologous recombinant vector into a skeleton vector containing a tetO element like the construction of a conventional vector; the positive rate of homologous recombination of mediated DNA large fragments (more than 2000 base pairs) can be improved; and the homologous recombination positive rate can be improved by 2-3 times in the preparation of a gene modified animal model.

Description

Method capable of improving homologous recombination efficiency of CRISPR/Cas9 system and application

Technical Field

The invention belongs to the technical field of bioengineering, and particularly belongs to the technical field of recombination.

Background

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) system is an adaptive immune defense mechanism formed in the long-term evolution process of bacteria and archaea, and is used for resisting invading viruses and exogenous DNA. The Cas9 nuclease of the CRISPR/Cas9 system can cause DNA double strand break aiming at a specific target site of a genome through the combination of a gRNA and a specific target sequence under the guidance of a single-stranded guide RNA (gRNA), thereby efficiently playing the gene editing role, and being an important tool for editing genomes of various organisms. In mammalian cells, CRISPR/Cas 9-initiated DNA double strand breaks can be repaired by at least two different competing mechanisms, namely non-homologous end joining (NHEJ) and homologous recombination-mediated targeted repair (HDR): NHEJ results in DNA insertions and deletions, while HDR can mediate precise genetic modifications. HDR repair can be artificially introduced to repair DNA templates, and the genome of an organism is artificially written, deleted and replaced at DNA fragmentation sites by taking the repair DNA templates as a standard. Therefore, HDR-mediated precise gene editing has great application prospects and applicability in basic research and clinical applications, such as construction of animal models for gene knock-in and gene therapy for correcting gene mutations.

Although HDR-mediated precise repair has a wide application prospect, HDR recombination efficiency is greatly limited. In response to this situation, scientists have studied to improve HDR efficiency from several aspects: 1) enhancement of HDR by inhibition of NHEJ or small molecules that up-regulate the HDR pathway; 2) the cell cycle process or time control of Cas9 expression is regulated through small molecules or Cas9 protein fusion, so that the HDR efficiency is improved; 3) the frequency of HDR occurrence is increased by increasing the local concentration of donor DNA at the double strand break site. Studies show that various small molecule compounds obtained by the methods 1) and 2) can improve HDR efficiency in cells and in vivo, but most of the compounds have negative effects on cell or embryo growth and inconsistent efficiency of different cell types or aiming at different sites, and meanwhile, the interference of small molecules on endogenous genes also has potential harmful side effects (Eric J. Aird, et al, incorporated Cas9-mediated homology-directed repair effective mutation and restriction of DNA repair template, COMMUNICATIONS BIOGY, (2018)1:54, DOI: 10.1038/s 03-420018-. Previous studies suggest that a key limiting step for HDR may be the local concentration of double strand break site donor DNA, and recently various laboratories have reported methods to improve HDR efficiency using the strategy of method 3): the Beijing university Jianzhong Jeff Xi laboratory and the American university of Wisconsin, Madison division Krishanu Saha laboratory utilize the binding activity of Biotin (Biotin) -Streptavidin (Streptavidin) to carry out Biotin modification on single-stranded DNA (ssDNA), and respectively construct Cas9-Avidin fusion protein and construct a gRNA linker capable of non-covalently binding with Streptavidin to increase the ssDNA concentration of the Cas9/gRNA complex locally at the DNA cleavage site, thereby increasing the HDR efficiency (Ming Ma, et. al, effective generation of microbial catalysis-colony-flow antibodies using the Cas 9-Avidin/Biotin-done DNA system. Research (2017) 27: 578. 10.1038/10.1038. J. 7. J. Pat. No. 7. 2017. see. branched. C. 12. branched RNA-peptide-binding RNA-peptide linker of nucleic acids, 8: 1711, DOI: 10.1038/s 41467-017-01875-9); the HDR efficiency was improved by O6-Benzylguanine (BG) modification of ssDNA by the Covalent binding of Cas9-SNAP fusion protein to BG-ssDNA at the university of Zurich, Switzerland, by using the Covalent binding of Cas9-SNAP fusion protein to BG-ssDNA (Natasa Savic, et al, equivalent linking of the DNA repalign to the CRISPR-Cas9 nucleic acids handling homology-directed repalign. eLife 2018; 7: e33761. DOI: https:// DOI. org/10.7554/eLife.333333338); the strategy that the HUH endonuclease can form covalent bonds with a specific sequence of unmodified single-stranded DNA (ssDNA) is utilized by the Wendy R. Gordon laboratory of Minnesota university, ssDNA is enriched to recombination sites through Cas9-HUH fusion protein, and HDR efficiency is improved (Eric J. Aird, et al, incorporated Cas9-mediated homology-directed repair efficiency genetic modification of DNA repair template, COMMUNICATIONSBIOLOGY, (2018)1:54, DOI 10.1038/s 42003-018-; the group of Tao Liu, university of Beijing, uses genetic code expansion techniques to chemically modify Cas9 proteins with azide-containing unnatural amino acids (ncAA) at specific positions of Cas9 proteins, which modifications allow for recruitment of the modified donor DNA template into the cleavage complex by alkyne-azide cycloaddition reactions followed by conjugation of Dibenzylcyclooctyne (DBCO) -containing donor ssODN or DNA adaptors, increasing the HDR efficiency (Xinyu Ling, et. al, improvement of the understanding of the affinity of the cleavage genome with site-specific Cas9-oligonucleotide conjugates, Science Advances 08 Apr 2020: Vol. 6, No. 15, eaaz0051, DOI: 10.1126/Science dv. aaz0051). These strategies have some limitations: 1) additional chemical modification and purification of ssDNA or Cas9 protein are often required, increasing the application cost; 2) the long ssDNA (more than 200 nucleotides in length) with modified ends has the supply problems of difficult preparation, high price and the like, and limits the application of recombination repair to large DNA fragments (more than 2000 base pairs); 3) the modification sites for ssDNA are limited, usually at one end of the ssDNA, and may not be optimal for HDR reaction efficiency.

Therefore, in order to improve the HDR efficiency mediated by CRISPR/Cas9, a new method which is simple to prepare and low in cost and can improve the HDR efficiency, especially the HDR efficiency of large-fragment DNA homologous recombination, is urgently needed to be developed.

Disclosure of Invention

Aiming at the problem of low HDR efficiency of the current CRISPR/Cas9 system, the invention describes a novel CRISPR/Cas9 mediated system for improving HDR homologous recombination efficiency, which mainly comprises three parts: cas9-TetR fusion protein, guide rna (grna), and donor DNA containing a tetracycline operator (tetO) element. Wherein, the Cas9-TetR fusion protein fuses Cas9 with a TetR (Tet reducer protein, TetR) structural domain through a linker; TetR is the protein domain of the regulatory gene expression repressor and tetO element binding in the tetracycline resistance operon of E.coli Tn 10; donor dna (donor vector) containing the tetracycline operator (tetO) element is a recombinant vector containing the tetO element. According to literature reports, the donor DNA concentration of the homologous sequence of the DNA break site may be a key rate-limiting step in the development of homologous recombination HDR. Thus, the inventive assumption of this system is that if the donor DNA is associated with the CRISPR/Cas9 module, there is more opportunity for the donor DNA to appear at the break site, then there is a greater probability that the donor DNA will be used.

In the invention, on one hand, the inventor obtains an artificial fusion protein Cas9-TetR through screening, and obtains a fusion protein which not only has high Cas9 protein nuclease activity, but also has the binding activity of a TetR structural domain to a tetO element by comparing the influence of connecting peptides with different lengths between Cas9 and TetR on the protein activity; on the other hand, the inventors compared the effects of different numbers of tetO elements and positions on the efficiency of homologous recombination, and screened out the information on the number and position of tetO elements having high efficiency of homologous recombination. After the Cas9-TetR protein is combined with the gRNA, the DNA at a specific site is cut under the guidance of the gRNA to cause double-strand DNA break, and the TetR domain of the Cas9-TetR can be combined with a tetO element on donor DNA to recruit more donor DNA to the DNA break site, so that the positive rate of homologous recombination HDR by using the donor DNA as a template is improved.

Specifically, the present invention relates to:

a fusion protein comprising a Cas9 protein and a TetR protein linked by a flexible linker peptide. The flexible linker peptide comprises 1 to 6 GGGGS domains, preferably 4 domains.

Further, the coding sequence of the Cas9 protein is shown as SEQ ID NO. 1, and the coding sequence of the TetR protein is shown as SEQ ID NO. 2.

Furthermore, the encoding nucleic acid sequence of the fusion protein is shown as SEQ ID NO. 13, the protein sequence is shown as SEQ ID NO. 14, and the fusion protein comprises deletion, substitution and homology of more than 90 percent on the basis of maintaining functions to a certain extent.

The invention also provides a homologous recombination system, which comprises Cas9-TetR fusion protein, guide RNA and a vector containing a tetracycline operator (tetO) element.

The tetO sites are positioned at the outer side of the homologous recombination left arm and/or the homologous recombination right arm of the recombination vector, and the number of the tetOs is 1-7 per arm.

Preferably, 3 tetO sites are added simultaneously outside the left arm of homologous recombination and outside the right arm of homologous recombination.

Further, the vector containing the tetracycline operator (tetO) element is a DNA vector; preferably, the vector comprises a homologous recombination left arm, an insertion element and a homologous recombination right arm which are connected in sequence from 5 'to 3'.

The insertion element is a gene that can be expressed and has a certain function, and specific selection can be found in the detailed description section.

Further, the system may include a guide RNA expression vector or Cas9-TetR expression vector or Cas9-TetR-gRNA expression vector.

The invention also provides a fusion protein and a homologous recombination system, and application of the fusion protein and the homologous recombination system in improving the homologous recombination positive rate, wherein the homologous recombination vector is a DNA fragment of more than 2000 bp.

Further, provides the application of the fusion protein and the homologous recombination system in preparing a gene modification animal model.

The present invention further relates to a gene recombination method comprising:

step 1: gene synthesis or plasmid template amplification mode to obtain homologous recombination left arm, homologous recombination right arm, and inserted element sequence segment, PCR amplification, and connecting the vector and the segment to form homologous recombination vector;

step 2: aiming at a gRNA sequence designed by a genome site of a target cell, a Cas9-tetR expression vector and a gRNA expression vector are constructed; or in vitro transcription to obtain Cas9-tetR mRNA, and artificially synthesizing gRNA;

and step 3: introducing the homologous recombination vector obtained in the step 1, the Cas9-tetR and gRNA expression vector obtained in the step 2 or the Cas9-tetR mRNA and artificially synthesized gRNA into a target cell together;

wherein the vector in step 1 is a plasmid vector comprising tetO.

Wherein, the introduction mode in the step 3 is microinjection or cotransfection.

Advantageous effects

Compared with the prior method, the method of the invention has the following advantages:

1) the recombinant vector or the Cas9 protein does not need to be subjected to additional chemical modification and purification, and the homologous recombinant vector is constructed into a skeleton vector containing a tetO element just like the construction of a conventional vector;

2) the positive rate of homologous recombination of mediated DNA large fragments (more than 2000 base pairs) can be improved;

3) the homologous recombination positive rate can be improved by 2-3 times in the preparation of a gene modified animal model.

Drawings

FIG. 1 shows a schematic diagram of a recombination enhancement system to increase the efficiency of homologous recombination; wherein: panel a in fig. 1 is a schematic representation of homologous recombination mediated by a conventional CRISPR/Cas9 system; panel B in figure 1 is a schematic representation of the recombination enhancement system mediated increase in efficiency of homologous recombination in which the Cas9-TetR fusion protein recruits the recombined donor DNA template to the periphery of the Cas9 cleavage site by binding to tetO elements located at both ends of the homology arm, increasing the efficiency of HDR generation.

Figure 2 shows Cas9 cleavage activity of Cas9 and 3 Cas9-tetR fusion proteins against EGFP; wherein: panel A in FIG. 2 is a schematic representation of a Cas9-tetR fusion protein structure; FIG. 2B is a schematic diagram of the principle of reflecting the cleavage activity of Cas9 and Cas9-tetR fusion protein Cas9 by using EGFP-expressing fluorescent cell lines; panel C in FIG. 2 is the flow cytometry results of the cleavage activity of Cas9, Cas9-GGGGS-TetR, Cas9- (GGGGS)2-TetR and Cas9- (GGGGS)4-TetR against EGFP, respectively, in CHO-K1-EGFP cell lines.

FIG. 3 shows the effect of the number and location of tetO on the efficiency of Cas9-tetR mediated homologous recombination HDR; wherein: FIG. 3A is schematic diagram of principle of reflecting efficiency of homologous recombination HDR by inserting IRES-tdTomato system in 3' UTR region of Actin gene by using CRISPR/Cas 9; FIG. 3B is a schematic diagram of 6 homologous recombination vectors with different amounts and positions of tetO added; FIG. 3 is a C-plot representing the flow cytometry detection results of the effect of single-sided different tetO number of recombinant vectors on the efficiency of IRES-tdTomato homologous recombination HDR; the D plot in FIG. 3 is the statistical analysis of the experiments for the C plot in FIG. 3 (. about.p < 0.05; p < 0.01; p < 0.001); FIG. 3 is a diagram E showing the flow cytometry detection results of the influence of the addition of 3 tetO elements to the single-side homology arm and the addition of 3 tetO elements to the outer side of the two-side homology arms on the efficiency of IRES-tdTomato homologous recombination HDR; the F plot in FIG. 3 is a statistical analysis of the experiment on the E plot in FIG. 3 (. about.p < 0.05; p < 0.01; p < 0.001).

FIG. 4 shows the difference in efficiency of insertion of the CAG-zsGreen-wpre-polyA fragment at the Rosa26 site in fertilized eggs using the Cas9 and Cas9-tetR systems; wherein: FIG. 4 is a schematic diagram of FIG. 4; FIG. 4B is a photograph showing the results of fluorescence photographs of 3.5-day embryos of each group; FIG. 4C is an electrophoretogram of PCR-identified homologous recombination for each group of embryos. The left panel shows the results of the identification of the left arm of homologous recombination, and the right panel shows the results of the right arm of homologous recombination (the DNA template in lane 1 is the wild-type mouse genome control; the DNA template in lane 2 is the blastocyst genome injected with only tetO-zsGreen recombinant vector; the DNA template in lane 3 is the blastocyst genome injected with Cas9/tetO-zsGreen recombinant vector; the DNA template in lane 4 is the blastocyst genome injected with Cas9-tetR/tetO-zsGreen recombinant vector; and M is 1kb DNA marker).

FIG. 5 shows the difference in efficiency of making a mouse with a Rosa26 site inserted into CAG-LSL-zsGreen-WPRE-polyA using Cas9 and Cas9-tetR systems; wherein: FIG. 5A is a schematic diagram of the preparation of mice conditionally overexpressed at the Rosa26 site; the B picture in FIG. 5 is a PCR electrophoretogram prepared by using Cas9 system to obtain genotype identification of F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The number is the mouse number, and M is 1kb DNA marker; panel C in figure 5 is a PCR electropherogram prepared using the Cas9-tetR system to obtain genotype identification for F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The numbers are mouse numbers and M is a 1kb DNA marker.

FIG. 6 shows the difference in efficiency of the preparation of H11 site inserted CAG-LSL-mTagBFP2-WPRE-poly conditional overexpression mice using Cas9 and Cas9-tetR systems; wherein: FIG. 6A is a schematic diagram of the preparation of mice conditionally overexpressed at H11 site; the B picture in FIG. 6 is a PCR electrophoretogram prepared by using Cas9 system to obtain genotype identification of F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The number is the mouse number, and M is 1kb DNA marker; panel C in figure 6 is a PCR electropherogram prepared using the Cas9-tetR system to obtain genotype identification for F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The numbers are mouse numbers and M is a 1kb DNA marker.

FIG. 7 shows the difference in efficiency of making Tpc1 conditional knockout mice using Cas9 and Cas9-tetR systems; wherein: panel A in FIG. 7 is a schematic diagram showing the principle of preparation of a TPC1 conditional knockout mouse (TPC 1 flox); the B picture in FIG. 7 is a PCR electrophoretogram prepared by using Cas9 system to obtain genotype identification of F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The number is the mouse number, and M is 1kb DNA marker; panel C in FIG. 7 is a PCR electropherogram prepared using the Cas9-tetR system to obtain genotype identification for F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The numbers are mouse numbers and M is a 1kb DNA marker.

FIG. 8 shows the difference in efficiency of Tmem119-2A-tdTomato mice prepared using Cas9 and Cas9-tetR systems; wherein: FIG. 8A is a schematic diagram of the preparation of Tmem119-2A-tdTomato mice; the B picture in FIG. 8 is a PCR electrophoretogram prepared by using Cas9 system to obtain genotype identification of F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The number is the mouse number, and M is 1kb DNA marker; panel C in FIG. 8 is a PCR electropherogram prepared using the Cas9-tetR system to obtain genotype identification for F0 mouse. The top panel shows the identification result of the left arm homologous recombination, and the bottom panel shows the identification result of the right arm homologous recombination. The numbers are mouse numbers and M is a 1kb DNA marker.

Detailed Description

Example one

Cas9 and Cas9-tetR series expression vector construction

The construction process of pcDNA3.1-Cas9, pcDNA3.1-Cas9-GGGGS-TetR, pcDNA3.1-Cas9- (GGGGS)2-TetR, and pcDNA3.1-Cas9- (GGGGS)4-TetR expression vector is as follows:

1) the Cas9 protein coding sequence and the TetR structural domain protein coding sequence are synthesized by the company of biological engineering (Shanghai), and the sequence information is SEQ ID NO: 1-2;

2) according to the insertion element sequences of a backbone vector sequence Cas9, a Cas9-GGGGS-TetR, a Cas9- (GGGGS)2-TetR and a Cas9- (GGGGGGS) 4-TetR of pcDNA3.1, PCR primers (the primer sequence information is shown In the following table, SEQ ID NO: 3-9) are respectively designed, a synthesized Cas9 and TetR fragment is used as a template for amplification to obtain a Cas9, a GGGGS-TetR, (GGGGGGS) 2-TetR and a (GGGGS)4-TetR fragment, and a pcDNA3.1-Cas9, a pcDNA3.1-Cas 9-GGS-TetR, a pcDNA3.1-9- (GGCas) 2-TetDNA, a pcDNA3.1-9- (GGS) 4-TetR, a vector is constructed by a fragment In-Fusion kit from Takara company, and the vector is correctly expressed after enzyme digestion and confirmation.

Example two

Comparison of cleavage activity of Cas9, Cas9-tetR fusion proteins against Cas9, a sequence of interest

The CHO-K1-EGFP cell strain constructed earlier by the inventor is used as a verification system for verifying the target sequence nuclease cleavage activity of 3 Cas9-tetR (the protein structure is shown as a diagram in figure 2), and the principle of the system is shown as a diagram B in figure 2. In previous work, the inventors inserted a CAG-EGFP-IRES-BSD-polyA expression cassette site-specifically at position CAAGTATACACTTGAGCCAGTAGTGGGGGGAGGGGTGG (SEQ ID NO:10) of the CHO-K1 cell line. By constructing a gRNA expression vector aiming at EGFP and cotransfecting a CHO-K1-EGFP cell strain with a Cas9 expression vector and 3 Cas9-tetR expression vectors, the Cas9 cuts an EGFP locus under the guidance of the gRNA to cause NHEJ, if the reading frame is shifted, the EGFP fluorescence disappears, and if the frame shift is not caused, the EGFP protein still expresses fluorescence. Because the probability that cleavage by Cas9 triggers reading frame shift of EGFP is the same, the ratio of EGFP fluorescence-depleted cells can be used as the basis for Cas9 activity.

Constructing an EGFP gRNA expression vector:

target sites for EGFP gRNA were: CGATGCCCTTCAGCTCGATGCGG (SEQ ID NO: 11, PAM sequence in bold underlined portion), the process for constructing EGFP gRNA expression vector was: a DNA fragment (see SEQ ID NO: 12) containing a U6 promoter, a gRNA target sequence, a gRNA scaffold and other expression elements is obtained through whole-gene synthesis, and is connected to a pUC57 vector through enzyme digestion, so that a gRNA expression vector aiming at the EGFP fragment is obtained.

Flow cytometry was used to compare the effect of 3 Cas9-tetR structures on Cas9 nuclease activity as follows:

1) using a Lipo3000 transfection reagent, operating according to instructions, and co-transfecting a Cas9/EGFP-gRNA expression vector, a Cas9-GGGGS-TetR/EGFP-gRNA expression vector, a Cas9- (GGGGS)2-TetR/EGFP-gRNA expression vector, a Cas9- (GGGGS)4-TetR/EGFP-gRNA expression vector with CHO-K1-EGFP cells respectively;

2) flow cytometry detection

After 6 days of transfection, old culture medium is removed from transfected cells, an appropriate amount of PBS is added for cleaning, after trypsinization, the cells are centrifugally collected, supernatant is removed, PBS is added for washing the cells for two times for resuspension, and then the cells are tested on a machine. Representative results of the assay are shown in panel C of figure 2. According to the analysis result: the fraction of EGFP-negative cells (17.5%) of Cas9-GGGGS-TetR group was significantly lower than Cas9 control group (33.2%), Cas9- (GGGGS)2-TetR group (28.2%) and Cas9- (GGGGS)4-TetR group (34.7%) were not significantly different compared to Cas9 control group. According to this result, the fusion of linker GGGGS-tetR in Cas9-GGGGS-tetR protein affects the nuclease activity of Cas9 itself; the fusion of Cas9- (GGGGS)4-TetR protein linker and TetR did not affect the nuclease activity of Cas 9. Therefore, Cas9- (GGGGS)4-TetR was selected as the Cas9-tetR fusion protein in the subsequent experiment, Cas9-tetR for short (the nucleic acid sequence is shown as SEQ ID NO: 13, and the protein sequence is shown as SEQ ID NO: 14).

EXAMPLE III

Effect of different tetO numbers and positions on Cas9-tetR mediated homologous recombination HDR efficiency

By inserting an IRES-tdTomato expression frame through homologous recombination in a 3' UTR region of an Actin gene of a Neuro2a cell line, the influence of different tetO numbers and positions on the HDR efficiency of homologous recombination mediated by Cas9- (GGGGS)4-TetR (hereinafter, abbreviated as "Cas 9-tetR") is evaluated through a detection system reflecting the HDR efficiency of homologous recombination by the proportion of tdTomato fluorescent cells, and the principle schematic diagram of the detection system is shown as a graph A in FIG. 3: in this system, the IRES-tdTomato element will express tdTomato fluorescence only if it is inserted into the transcribed region of the gene; actin is a housekeeping gene and is continuously and highly expressed in cells, gRNA is designed aiming at the 3 'UTR (untranslated regions) region of Actin, double-stranded DNA breakage is caused by Cas9 or Cas9-tetR, an IRES-tdTomato element is integrated into the 3' UTR region of Actin through homologous recombination HDR, IRES-tdTomato is transcribed and translated into red fluorescent protein, and cells show red color. Although IRES-tdTomato elements may also be randomly inserted into the transcription regions of other genes, resulting in tdTomato expression, the probability is overall low, and thus the efficiency of homologous recombination HDR can be reflected by the amount of red fluorescence of tdTomato.

gRNA target sites for the Actin 3' UTR were: CCACCCCCACTCCTAAGAGGAGG (PAM sequence underlined in bold, SEQ ID NO: 15), a gRNA expression vector was constructed for this site.

Construction of IRES-tdTomato recombinant vector containing tetO site

To verify the effect of the number and location of tetO sites on the efficiency of Cas9-tetR mediated homologous recombination HDR, recombination vectors containing 0, 1, 2, 3, 4, and 7 tetO sites at the 5' end of the left arm of homologous recombination (named donor, donor-L1, donor-L2, donor-L3, donor-L4, and donor-L7, respectively) were constructed as shown in Panel B of FIG. 3. The sequence information of 1, 2, 3, 4 and 7 tetO sites are SEQ ID NO: 16-20. The sequence of homologous recombination fragments of the recombinant vector is SEQ ID NO: 21, wherein the sequence of the homologous recombination left arm is SEQ ID NO. 22, the sequence of the homologous recombination right arm is SEQ ID NO. 23, and the sequence of the insertion element IRES-tdTomato is SEQ ID NO. 24.

The experimental procedure for comparing the effect of recombinant vectors containing different tetO number sites on the efficiency of homologous recombination HDR by flow cytometry is as follows:

1) by using a Lipo3000 transfection reagent, operating according to the instruction, simultaneously co-transfecting a Cas9-tetR expression vector and a gRNA expression vector with a donor-L1 recombinant vector, a donor-L2 recombinant vector, a donor-L3 recombinant vector, a donor-L4 recombinant vector and a donor-L7 recombinant vector respectively to a Neuro2a cell line of a mouse, and performing efficiency comparison with a Neuro2a group co-transfected by a Cas9 expression vector, a gRNA expression vector and a donor recombinant vector;

2) flow cytometry detection

After 72 hours of transfection, old culture medium is removed from transfected cells, a proper amount of PBS is added for cleaning, after trypsinization, the cells are centrifugally collected, supernatant is removed, PBS is added for washing the cells for two times, and after resuspension, the cells are tested on a machine. Representative results of the assay are shown in graph C in fig. 3, and statistical analysis results are shown in graph D in fig. 3. According to the analysis result: the Cas9-tetR/tetO-donor system can significantly improve the efficiency of homologous recombination in cells, with the addition of 3 tetO sites at the 5' end of the homologous left arm having the highest efficiency of homologous recombination compared to the number of other tetO sites. To further analyze whether the one-sided and two-sided tetO sites have an effect on the efficiency of homologous recombination, the inventors further constructed a recombination vector (donor-LR 3) containing 3 tetO sites outside the left and right arms of homologous recombination. The detection method and the process for comparing the same influence of the recombination vectors containing different tetO number sites on the homologous recombination HDR efficiency through the flow cytometry are compared, and the homologous recombination HDR efficiency is compared by independently adding 3 tetOs outside the homologous left arm and simultaneously adding 3 tetOs outside the homologous left arm and the homologous right arm through the flow cytometry. Representative results of flow cytometry assays are shown in FIG. 3, panel E, and statistical analysis in FIG. 3, panel F. According to the analysis result: compared with the method of adding 3 tetOs outside the homologous left arm, the method of simultaneously adding 3 tetOs outside the homologous left arm and the homologous right arm has higher homologous recombination efficiency, and in subsequent verification, 3 tetO elements are simultaneously added outside the homologous left arm and the homologous right arm to serve as homologous recombination vectors.

Example four

Comparison of efficiency of Cas9 and Cas9-tetR systems in mediating homologous recombination in mouse zygotes

The CAG-zsGreen-WPRE-polyA (zsGreen) expression frame is integrated to a mouse genome Rosa26 site by a Cas9 and Cas9-tetR system respectively through a homologous recombination mode, after the zsGreen expression frame is inserted, a CAG promoter can drive the expression of zsGreen fluorescence, the generation efficiency of homologous recombination HDR can be determined through the expression reaction of the fluorescence, the strategy schematic diagram is shown as an A diagram in figure 4, and the flow is as follows:

1) constructing a tetO-zsGreen homologous recombination vector: obtaining homologous recombination left arm (SEQ ID NO: 25), right arm (SEQ ID NO: 26) and zsGreen-WPRE-polyA element (SEQ ID NO: 27) through DNA synthesis, obtaining a required fragment for vector construction through PCR amplification, obtaining a CAG promoter fragment (SEQ ID NO: 28) through amplification by taking plvct-tTR-KRAB plasmid as a template, and then inserting 4 fragments of the homologous recombination left arm, the CAG promoter, the zsGreen-WPRE-polyA and the homologous recombination right arm into a skeleton vector (PBR 322-tetO, SEQ ID NO: 29) through an In-Fusion method to construct a tetO-zsGreen homologous recombination vector;

2) cas9 and Cas9-tetR mRNA are obtained by in vitro transcription, wherein a Cas9 and Cas9-tetR expression vector constructed in the previous period is used as a template, a T7 promoter is used as a part of a primer sequence to be amplified to obtain a T7-Cas9-polyA fragment and a T7-Cas9-tetR-polyA fragment, the T7-Cas9-polyA fragment and the T7-Cas9-tetR-polyA fragment are used as DNA templates for in vitro transcription, and the Cas9 mRNA and the Cas9-tetR mRNA are obtained by in vitro transcription according to the guidance of a T7 in vitro transcription kit instruction (purchased from Life technologies). Artificially synthesizing gRNA (Rosa 26-gRNA for short) aiming at the Rosa26 locus, wherein the sequence is shown as SEQ ID NO: 30;

3) injecting the fertilized eggs: fertilized eggs from C57BL/6 mice were divided into three groups as shown in the following table:

grouping	Cas9 mRNA（ng/ul）	Cas9-tetR mRNA (ng/ul）	Rosa26-gRNA (ng/ul）	tetO-zsGreen plasmid (ng/ul)
					Control group	0	0	0	5
Experimental group 1	5	0	2	5
					Experimental group 2	0	5	2	5

Microinjection of fertilized eggs was performed according to the method in the handbook of mouse embryo manipulation experiments (third edition), and only tetO-zsGreen recombinant vector was injected for the control group without Cas9 or Cas 9-tetR; experimental group 1 injection of a mixture of Cas 9/Rosa 26-RNA/tetO-zsGreen; experiment group 2 injected with a mixture of Cas9-tetR/Rosa26-RNA/tetO-zsGreen, the injected fertilized eggs were transferred to an incubator and cultured for 3 days, fluorescence photographing was performed, the fluorescence expression of zsGreen was detected (FIG. 4, panel B), and after the fluorescence embryos were cleaved, whether the zGreen fragments were integrated into the Rosa sites by homologous recombination was identified by PCR.

Identifying primer sequence information:

primer name	Primer sequence (5' ->3')
		Left arm forward primer	TGTGTTGTGTGTGCTTGTGC (SEQ ID NO：31)
Left arm reverse primer	CAATCCAGCGGACCTTCCTT (SEQ ID NO：32)
		Right arm forward primer	AGAGTGAAGCAGAACGTGGG (SEQ ID NO：33)
Right arm reverse primer	AGACCTGTTGCTGCTCAGAC (SEQ ID NO：34)

And (3) PCR reaction system:

PCR reaction composition	Volume (mu l)
		ddH2O	31
PCR Buffer	10
		2.5 mM dNTP	4
Forward primer	1
		Reverse primer	1
DNA Polymerase	2
		genomic DNA	1
Total of	50

PCR reaction procedure:

step (ii) of	Temperature (. degree.C.)	Time	Remarks for note
				1	98	2 min
2	98	15 sec
				3	68	15 sec
4	68	1 min	Repeat steps 2-4 for a total of 34 cycles
				5	68	1 min
6	12	10 min

The results of PCR identification electrophoresis are shown in FIG. 4C. The result of fluorescence of the blastocyst and the result of identification of homologous recombination show that: simply injecting a tetO-zsGreen recombinant vector group, detecting that 2 blastula have zsGreen fluorescence (the fluorescence positive rate is 1.9%), taking the fluorescent blastula for PCR identification, failing to amplify a positive PCR product for homologous recombination, and randomly inserting the detected fluorescent embryos into the recombinant vector; cas9/tetO-zsGreen recombinant vector group is injected, zsGreen fluorescence (the fluorescence positive rate is 4.4%) is detected in 4 blastula, the fluorescent blastula is taken for PCR identification, a positive PCR product for homologous recombination can be amplified, and the detected fluorescent embryos are subjected to homologous recombination; cas9-tetR/tetO-zsGreen recombinant vector group is injected, zsGreen fluorescence (fluorescence positive rate is 9.4%) is detected in 11 blastula, the fluorescent blastula is taken for PCR identification, a positive PCR product for homologous recombination can be amplified, and the detected fluorescent embryos are subjected to homologous recombination.

In conclusion, by using both the Cas9 system and the Cas9-tetR system, the integration of the exogenous fragment of CAG-zsGreen-WPRE-polyA about 3.3kb into the Rosa26 site through homologous recombination can be realized, and preliminary evidence shows that the Cas9-tetR system has higher efficiency of mediating homologous recombination HDR than the conventional Cas9 system.

EXAMPLE five

Cas9 and Cas9-tetR System for comparison of efficiency in making mice with knockin of Rosa26 site Gene

A homologous recombination scheme for preparing a Rosa26-CAG-LSL-zsGreen-WPRE-polyA (R26-LSL-zsGreen) fixed-point knock-in mouse by using a Cas9 system and a Cas9-tetR system is shown as a diagram A in figure 5, and the flow is as follows:

1) constructing a Rosa26 site tetO-CAG-LSL-zsGreen-WPRE-polyA homologous recombination vector: obtaining a loxp-stop-loxp sequence (LSL for short, SEQ ID NO:35) through whole gene synthesis, connecting the LSL sequence with a CAG-zsGreen-WPRE-polyA recombinant vector through enzyme digestion on the CAG-zsGreen-WPRE-polyA recombinant vector obtained by the previous construction, and constructing to obtain a tetO-CAG-LSL-zsGreen-WPRE-polyA homologous recombinant vector (LSL-zsGreen for short);

2) injecting the fertilized eggs: fertilized eggs of C57BL/6 mice were collected and divided into two groups: carrying out microinjection on fertilized eggs according to a method in a mouse embryo operation experimental manual (third edition) in the Cas9 group and the Cas9-tetR group, wherein the injection dosage is the same as that in the fourth example, the injected fertilized eggs are transplanted to an oviduct of a receptor mother mouse after being cultured in a culture box for a short time, and a genetically modified mouse F0 generation mouse is obtained;

3) mouse genotype identification: whether the CAG-LSL-zsGreen-WPRE-polyA fragment of the F0 generation mouse is integrated to the target Rosa26 site through homologous recombination or not is identified by respectively designing primers on the outside of the homologous arm and the insertion element CAG-LSL-zsGreen-WPRE-polyA for PCR amplification, and the identification primer and the PCR condition are the same as the fourth embodiment.

The electrophoresis results of the homologous recombination PCR identification are shown in the B diagram in FIG. 5 and the C diagram in FIG. 5 (the positive mice in the B diagram in FIG. 5 are No. 5, 9, 12; the positive mice in the C diagram in FIG. 5 are No. 2, 3, 7, 8, 11, 12, 13. the upper diagram is the identification result of the left arm homologous recombination, and the lower diagram is the identification result of the right arm homologous recombination), and the statistical results of the gene knock-in prepared by using the Cas9 system and the Cas9-tetR system are shown in the following table:

group of	Number of fertilized eggs transplanted	Number of mice born	Number of positive mice	Positive rate
					Cas9
	100	14	3	3%
					Cas9-tetR	100	17	7	7%

According to the result, a mouse model of the Rosa26 site-directed knock-in is prepared by using a Cas9-tetR system, a CAG-LSL-zsGreen-WPRE-polyA fragment of about 5.1kb is knocked in, and F0 generation mice have higher homologous recombination positive rate than that of a conventional Cas9 system.

EXAMPLE six

Cas9 and Cas9-tetR System for making H11 site knock-in mice

A homologously recombined sketch map of a H11-CAG-LSL-mTagBFP2-WPRE-polyA (called H11-LSL-mTagBFP2 for short) prepared by a Cas9 and Cas9-tetR system is shown in a diagram A in figure 6, and the specific construction flow is as follows:

1) constructing a H11 site tetO-CAG-LSL-mTagBFP2-WPRE-polyA homologous recombination vector: h11 site homologous recombination left arm (SEQ ID NO: 36), homologous recombination right arm (SEQ ID NO: 37) and mTagBFP2 sequence (SEQ ID NO:38) are obtained through whole gene synthesis, a previously constructed LSL-zsGreen vector is used as a template, a CAG-LSL and WPRE-polyA sequence are obtained through PCR amplification, and an H11 site tetO-CAG-LSL-mTagBFP2-WPRE-polyA homologous recombination vector (LSL-mTagBFP 2 for short) is finally constructed through an In-Fusion method;

2) injecting the fertilized eggs: fertilized eggs of C57BL/6 mice were collected and divided into two groups: carrying out microinjection on fertilized eggs according to a method in a mouse embryo operation experimental manual (third edition) in the Cas9 group and the Cas9-tetR group, wherein the injection dosage is the same as that in the fourth example, the injected fertilized eggs are transplanted to an oviduct of a receptor mother mouse after being cultured in a culture box for a short time, and a genetically modified mouse F0 generation mouse is obtained;

3) mouse genotype identification: whether the F0 generation mouse CAG-LSL-mTagBFP2-WPRE-polyA fragment is integrated to the target H11 site through homologous recombination or not is identified by utilizing a method of designing primers on the outside of the homologous arm and the insertion element CAG-LSL-mTagBFP2-WPRE-polyA respectively for PCR amplification, and the identification primers and PCR conditions are shown as follows.

PCR identification primer sequence information:

primer name	Primer sequence (5' ->3')
		Left arm forward primer	TGCATCCTCCAGGCTCTAGT (SEQ ID NO:39)
Left arm reverse primer	AGAGTGAAGCAGAACGTGGG (SEQ ID NO:40)
		Right arm forward primer	CAATCCAGCGGACCTTCCTT (SEQ ID NO:41)
Right arm reverse primer	CACAACCTGGTCAGTCCCTC (SEQ ID NO:42)

And (3) PCR reaction system:

PCR reaction composition	Volume (mu l)
		ddH2O	31
PCR Buffer	10
		2.5 mM dNTP	4
Forward primer	1
		Reverse primer	1
DNA Polymerase	2
		genomic DNA	1
Total of	50

PCR reaction procedure:

step (ii) of	Temperature (. degree.C.)	Time	Remarks for note
				1	98	2 min
2	98	15 sec
				3	68	15 sec
4	68	1 min	Repeat steps 2-4 for a total of 34 cycles
				5	68	1 min
6	12	10 min

The electrophoresis results of the homologous recombination PCR identification are shown in B diagram in FIG. 6 and C diagram in FIG. 6, and the right arm correct homologous recombination should amplify PCR product with 1.9kb size, and the right arm correct homologous recombination should amplify PCR product with 1.4kb size. (the B picture in figure 6 is the identification result of the Cas9 injection group homologous recombination mouse, the number of the positive mouse is 3, 9; the C picture in figure 6 is the identification result of the Cas9-tetR injection group homologous recombination mouse, the number of the positive mouse is 2, 4, 5, 10; the upper pictures of the B picture in figure 6 and the C picture in figure 6 are the identification result of the left arm homologous recombination, the lower picture is the identification result of the right arm homologous recombination, the upper figure of the pictures is the mouse number, and M is 1kb DNA marker).

Statistical results for generating gene knockins using the Cas9 system and Cas9-tetR system are shown in the following table:

group of	Number of fertilized eggs transplanted	Number of mice born	Number of positive mice	Positive rate
					Cas9
	100	12	2	2%
					Cas9-tetR	100	14	4	4%

The result shows that a H11 site-directed knock-in mouse model is prepared by using a Cas9-tetR system, a CAG-LSL-mTagBFP2-WPRE-polyA fragment of about 5.3kb is knocked in, and F0 generation mice have higher homologous recombination positive rate than the conventional Cas9 system.

EXAMPLE seven

Comparison of efficiency of Cas9 and Cas9-tetR System in the preparation of Tcp1 conditional knockout mice

A Tcp1 conditional knockout mouse (abbreviated as 'Tcp 1 flox') is prepared by respectively utilizing a Cas9 system and a Cas9-tetR system, a homologous recombination schematic diagram for preparing the mouse is shown as a diagram A in figure 7, and the specific construction flow is as follows:

1) constructing a Tcp1 flox homologous recombinant vector: obtaining a homologous recombination fragment through whole gene synthesis, wherein the fragment comprises a homologous recombination left arm (SEQ ID NO: 43), two loxp sites, a flox region (SEQ ID NO: 44) and a homologous recombination right arm (SEQ ID NO: 45), inserting the homologous recombination fragment into a PBR322-tetO framework vector through an In-Fusion method, and finally constructing to obtain a Tcp1 flox homologous recombination vector after correct sequencing;

2) injecting the fertilized eggs: fertilized eggs of C57BL/6 mice were collected and divided into two groups: carrying out microinjection on fertilized eggs according to a method in a mouse embryo operation experimental manual (third edition) in the Cas9 group and the Cas9-tetR group, wherein the injection dosage is the same as that in the fourth example, the injected fertilized eggs are transplanted to an oviduct of a receptor mother mouse after being cultured in a culture box for a short time, and a genetically modified mouse F0 generation mouse is obtained;

3) mouse genotype identification: whether F0 generation mice mediated the integration of flox region by homologous recombination was identified by PCR amplification using primers designed outside the homology arm and on the recombination sequence, the identifying primers and PCR conditions are shown in the following table:

PCR primer information:

primer name	Primer sequence (5' ->3')
		Left arm forward primer	ATCCCTACCGCTTTCCCGGCA (SEQ ID NO:46)
Left arm reverse primer	TCATCAATGCCACCAGTGGTTA (SEQ ID NO:47)
		Right arm forward primer	CAACCTGGCCGTTAAGATGC (SEQ ID NO:48)
Right arm reverse primer	CGATAGCCACTAATAACTGAT (SEQ ID NO:49)

And (3) PCR reaction system:

PCR reaction composition	Volume (mu l)
		ddH2O	31
PCR Buffer	10
		2.5 mM dNTP	4
Forward primer	1
		Reverse primer	1
DNA Polymerase	2
		genomic DNA	1
Total of	50

PCR reaction procedure:

step (ii) of	Temperature (. degree.C.)	Time	Remarks for note
				1	98	2 min
2	98	15 sec
				3	68	15 sec
4	68	1 min	Repeat steps 2-4 for a total of 34 cycles
				5	68	1 min
6	12	10 min

The electrophoresis results of the homologous recombination PCR identification are shown in B diagram in FIG. 7 and C diagram in FIG. 7, and the right arm correct homologous recombination should amplify a PCR product of 2.2kb in size. (the B picture in figure 7 is the identification result of the Cas9 injection group homologous recombination mouse, the number of the positive mouse is 6, 7 and 14; the C picture in figure 7 is the identification result of the Cas9-tetR injection group homologous recombination mouse, the number of the positive mouse is 3, 4, 10, 11 and 12; the B picture in figure 7 and the upper picture of the C picture in figure 7 are the identification result of the left arm homologous recombination, the lower picture is the identification result of the right arm homologous recombination, the upper figure of the picture is the mouse number, and M is 1kb DNA marker).

Statistics of the positivity of Tcp1 flox mice prepared using the Cas9 system and the Cas9-tetR system are shown in the following table:

group of	Number of fertilized eggs transplanted	Number of mice born	Number of positive mice	Positive rate
					Cas9
	100	20	3	3%
					Cas9-tetR	100	17	5	5%

The result shows that when a Tcp1 flox mouse model is prepared by using a Cas9-tetR system, the flox region is about 1kb, and F0 generation mice have higher homologous recombination positive rate than the conventional Cas9 system.

Example eight

Comparison of efficiency of Cas9 and Cas9-tetR System in preparation of Tmem119-tdTomato knock-in mice

A Tmem119-tdTomato knock-in mouse is prepared by respectively utilizing a Cas9 system and a Cas9-tetR system, a homologous recombination scheme for preparing the mouse is shown as a diagram A in figure 8, and the specific construction flow is as follows:

1) constructing a Tmem119-tdTomato homologous recombination vector: obtaining a homologous recombination fragment through whole gene synthesis, wherein the fragment comprises a homologous recombination left arm (SEQ ID NO: 50), a 2A-tdTomato element (SEQ ID NO: 51) and a homologous recombination right arm (SEQ ID NO: 52), inserting the homologous recombination fragment into a PBR322-tetO skeleton vector through an In-Fusion method, and finally constructing and obtaining a Tmem119-tdTomato homologous recombination vector after correct sequencing;

2) injecting the fertilized eggs: fertilized eggs of C57BL/6 mice were collected and divided into two groups: carrying out microinjection on fertilized eggs according to a method in a mouse embryo operation experimental manual (third edition) in the Cas9 group and the Cas9-tetR group, wherein the injection dosage is the same as that in the fourth example, the injected fertilized eggs are transplanted to an oviduct of a receptor mother mouse after being cultured in a culture box for a short time, and a genetically modified mouse F0 generation mouse is obtained;

3) mouse genotype identification: whether F0 generation mice mediated the integration of 2A-tdTomato element at the Tmem119 gene position by homologous recombination was identified by PCR amplification method using primers designed on the outside of the homology arm and the insertion element respectively, and the identification primers and PCR conditions are shown in the following table:

primer sequence information:

primer name	Primer sequence (5' ->3')
		Left arm forward primer	ATAACCTATCCCCTAACACATAAGCACCAG (SEQ ID NO:53)
Left arm reverse primer	CAGCTTGGTGTCCACGTAGT (SEQ ID NO:54)
		Right arm forward primer	CATGGACGAGCTGTACAAGTAA (SEQ ID NO:55 )
Right arm reverse primer	TGGAATGGTATTTGGGTGTT (SEQ ID NO:56)

And (3) PCR reaction system:

PCR reaction composition	Volume (mu l)
		ddH2O	31
PCR Buffer	10
		2.5 mM dNTP	4
Forward primer	1
		Reverse primer	1
DNA Polymerase	2
		genomic DNA	1
Total of	50

PCR reaction procedure:

step (ii) of	Temperature (. degree.C.)	Time	Remarks for note
				1	98	2 min
2	98	15 sec
				3	68	15 sec
4	68	1 min	Repeat steps 2-4 for a total of 34 cycles
				5	68	1 min
6	12	10 min

The electrophoresis results of the homologous recombination PCR identification are shown in B diagram in FIG. 8 and C diagram in FIG. 8, and the right arm correct homologous recombination should amplify a PCR product of 2.1 kb in size, and the right arm correct homologous recombination should amplify a PCR product of 1.9kb in size. (the B picture in figure 8 is the identification result of the Cas9 injection group homologous recombination mouse, the number of the positive mouse is 9; the C picture in figure 8 is the identification result of the Cas9-tetR injection group homologous recombination mouse, the number of the positive mouse is 1, 5, 11; the B picture in figure 8 and the C picture in figure 8 are the identification result of the left arm homologous recombination, the lower picture is the identification result of the right arm homologous recombination, the number above the picture is the mouse number, and M is 1kb DNA marker).

Statistical results for Tmem119-tdTomato mouse positivity prepared using the Cas9 system and the Cas9-tetR system are shown in the following table:

group of	Number of fertilized eggs transplanted	Number of mice born	Number of positive mice	Positive rate
					Cas9
	100	22	1	1%
					Cas9-tetR	100	15	3	3%

The result shows that Tmem119-tdTomato mouse model is prepared by using Cas9-tetR system, a 1.5kb DNA fragment is inserted, and F0 generation mice have higher homologous recombination positive rate than the conventional Cas9 system.

To sum up, the inventors screened an artificial fusion protein Cas9-TetR, which has the nuclease activity of Cas9 protein; the inventor screens and obtains the quantity and position information of tetO elements with high homologous recombination efficiency, and establishes a Cas9-TetR/tetO recombination enhancement system. The inventor uses the system to prepare a conditional overexpression mouse model and a conditional knockout mouse model and a gene knock-in mouse model of an unconventional locus at the conventional Rosa26 and H11 loci respectively, and compares the efficiency of the system and the efficiency of the conventional Cas9 system in the preparation process, and the result shows that the system can remarkably improve the homologous recombination efficiency by 2-3 times in the preparation process of a gene modification animal model.

Sequence listing

<110> Shanghai's Square model Biotech Co., Ltd

Shanghai Yushi Biological Technology Co., Ltd.

Guangdong Nanmo Biological Technology Co., Ltd.

<120> method capable of improving homologous recombination efficiency of CRISPR/Cas9 system and application

<160> 56

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4203

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag 60

aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 120

gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 180

aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 240

ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 300

gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 360

tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 420

gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 480

gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 540

ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 600

ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 660

agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 720

aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 780

ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 840

ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 900

gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 960

gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 1020

agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 1080

cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 1140

gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 1200

gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 1260

accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 1320

cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 1380

ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 1440

tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 1500

aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 1560

aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 1620

ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 1680

cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 1740

ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 1800

gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 1860

aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 1920

acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 1980

gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 2040

cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 2100

aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 2160

tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 2220

attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 2280

gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 2340

gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 2400

atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 2460

acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 2520

gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 2580

cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 2640

cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 2700

cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 2760

gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 2820

acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 2880

gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 2940

tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 3000

gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 3060

ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 3120

aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 3180

aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 3240

gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 3300

cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 3360

ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 3420

aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 3480

ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 3540

ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 3600

gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 3660

ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 3720

aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 3780

aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 3840

cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 3900

gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 3960

gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 4020

ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 4080

gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 4140

cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 4200

aag 4203

<210> 2

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60

ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120

ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180

gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240

aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300

ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360

tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420

actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480

cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540

ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600

cttaaatgtg aaagtgggtc gccaaaaaag aagagaaagg tc 642

<210> 3

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agggagaccc aagctgccac catgatggcc ccaaagaaga a 41

<210> 4

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctcggtacc aagctctact ttttcttttt tgcct 35

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

actgccaccg ccaccctttt tcttttttgc ctggc 35

<210> 6

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtggcggtg gcagtatggc tagattagat aaaag 35

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctcggtacc aagctttaga cctttctctt ctttt 35

<210> 8

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggtggcggtg gcagtggtgg cggtggcagt atggctagat tagataaaag 50

<210> 9

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggtggcggtg gcagtggtgg cggtggcagt ggtggcggtg gcagtggtgg cggtggcagt 60

atggctagat tagataaaag 80

<210> 10

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caagtataca cttgagccag tagtgggggg aggggtgg 38

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgatgccctt cagctcgatg cgg 23

<210> 12

<211> 491

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggatcccgac acgagggcct atttcccatg attccttcat atttgcatat acgatacaag 60

gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat 120

acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa 180

atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc 240

ttgtggaaag gacgaaacac cgcgatgccc ttcagctcga tggttttaga gctagaaata 300

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 360

ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc 420

gccaattctg cagacaaatg gagaggcatg tgacacgggc ccgtcgactg cagaggcctg 480

catgcaagct t 491

<210> 13

<211> 4908

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag 60

aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 120

gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 180

aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 240

ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 300

gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 360

tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 420

gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 480

gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 540

ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 600

ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 660

agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 720

aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 780

ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 840

ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 900

gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 960

gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 1020

agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 1080

cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 1140

gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 1200

gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 1260

accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 1320

cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 1380

ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 1440

tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 1500

aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 1560

aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 1620

ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 1680

cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 1740

ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 1800

gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 1860

aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 1920

acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 1980

gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 2040

cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 2100

aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 2160

tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 2220

attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 2280

gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 2340

gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 2400

atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 2460

acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 2520

gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 2580

cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 2640

cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 2700

cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 2760

gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 2820

acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 2880

gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 2940

tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 3000

gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 3060

ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 3120

aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 3180

aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 3240

gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 3300

cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 3360

ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 3420

aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 3480

ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 3540

ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 3600

gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 3660

ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 3720

aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 3780

aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 3840

cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 3900

gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 3960

gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 4020

ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 4080

gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 4140

cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 4200

aagggtggcg gtggcagtgg tggcggtggc agtggtggcg gtggcagtgg tggcggtggc 4260

agtatggcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag 4320

gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct 4380

acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg 4440

ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta 4500

cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta 4560

catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt 4620

ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat 4680

tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa 4740

acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac 4800

caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa 4860

caacttaaat gtgaaagtgg gtcgccaaaa aagaagagaa aggtctaa 4908

<210> 14

<211> 1635

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Met Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro

1 5 10 15

Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser

20 25 30

Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys

35 40 45

Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu

50 55 60

Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg

65 70 75 80

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

85 90 95

Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp

100 105 110

Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys

115 120 125

Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala

130 135 140

Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val

145 150 155 160

Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala

165 170 175

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

180 185 190

Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr

195 200 205

Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp

210 215 220

Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu

225 230 235 240

Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly

245 250 255

Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn

260 265 270

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

275 280 285

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

290 295 300

Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser

305 310 315 320

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

325 330 335

Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu

340 345 350

Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe

355 360 365

Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala

370 375 380

Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met

385 390 395 400

Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu

405 410 415

Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His

420 425 430

Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro

435 440 445

Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg

450 455 460

Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala

465 470 475 480

Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu

485 490 495

Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met

500 505 510

Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His

515 520 525

Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val

530 535 540

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

545 550 555 560

Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val

565 570 575

Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe

580 585 590

Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu

595 600 605

Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu

610 615 620

Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu

625 630 635 640

Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr

645 650 655

Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg

660 665 670

Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg

675 680 685

Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly

690 695 700

Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr

705 710 715 720

Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser

725 730 735

Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys

740 745 750

Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met

755 760 765

Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn

770 775 780

Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg

785 790 795 800

Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His

805 810 815

Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr

820 825 830

Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn

835 840 845

Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu

850 855 860

Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn

865 870 875 880

Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met

885 890 895

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

900 905 910

Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu

915 920 925

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

930 935 940

Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr

945 950 955 960

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

965 970 975

Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val

980 985 990

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

995 1000 1005

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

1010 1015 1020

Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1025 1030 1035 1040

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr

1045 1050 1055

Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly

1060 1065 1070

Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu

1075 1080 1085

Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu

1090 1095 1100

Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly

1105 1110 1115 1120

Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu

1125 1130 1135

Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp

1140 1145 1150

Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys

1155 1160 1165

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

1170 1175 1180

Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1185 1190 1195 1200

Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro

1205 1210 1215

Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala

1220 1225 1230

Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys

1235 1240 1245

Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly

1250 1255 1260

Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1265 1270 1275 1280

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg

1285 1290 1295

Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn

1300 1305 1310

Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His

1315 1320 1325

Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe

1330 1335 1340

Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu

1345 1350 1355 1360

Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg

1365 1370 1375

Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys

1380 1385 1390

Lys Ala Gly Gln Ala Lys Lys Lys Lys Gly Gly Gly Gly Ser Gly Gly

1395 1400 1405

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Arg

1410 1415 1420

Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu

1425 1430 1435 1440

Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly

1445 1450 1455

Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu

1460 1465 1470

Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe

1475 1480 1485

Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala

1490 1495 1500

Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val

1505 1510 1515 1520

His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn

1525 1530 1535

Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu

1540 1545 1550

Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu

1555 1560 1565

Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr

1570 1575 1580

Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His

1585 1590 1595 1600

Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys

1605 1610 1615

Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Pro Lys Lys Lys

1620 1625 1630

Arg Lys Val

1635

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccacccccac tcctaagagg agg 23

<210> 16

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gagtttacca ctccctatca gtgatagaga aaagtgaaag tc 42

<210> 17

<211> 84

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 60

cagtgataga gaaaagtgaa agtc 84

<210> 18

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 60

cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg 120

aaagtc 126

<210> 19

<211> 168

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 60

cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg 120

aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtc 168

<210> 20

<211> 294

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 60

cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg 120

aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact 180

ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga 240

aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa agtc 294

<210> 21

<211> 3619

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cctgaggctc ttttccagcc ttccttcttg ggtaagttgt agcctagtcc tttctccatc 60

taaaggtgac aaaactcctg aggccatagt acaagttaag tctgatttct gtcactcttc 120

tcttaggtat ggaatcctgt ggcatccatg aaactacatt caattccatc atgaagtgtg 180

acgttgacat ccgtaaagac ctctatgcca acacagtgct gtctggtggt accaccatgt 240

acccaggcat tgctgacagg atgcagaagg agattactgc tctggctcct agcaccatga 300

agatcaaggt aagctaagca tccttagctt ggtgagggtg ggccctgtgg ttgtcagagc 360

aaccttctag gtttaagggg aatcccagca cccagagagc tcaccattca ccatcttgtc 420

ttgctttctt cagatcattg ctcctcctga gcgcaagtac tctgtgtgga tcggtggctc 480

catcctggcc tcactgtcca ccttccagca gatgtggatc agcaagcagg agtacgatga 540

gtccggcccc tccatcgtgc accgcaagtg cttctaggcg gactgttact gagctgcgtt 600

ttacaccctt tctttgacaa aacctaactt gcgcagaaaa aaaaaaaata agagacaaca 660

ttggcatggc tttgtttttt taaatttttt ttaaagtttt tttttttttt tttttttttt 720

tttttaagtt tttttgtttt gttttggcgc ttttgactca ggatttaaaa actggaacgg 780

tgaaggcgac agcagttggt gcccctctcc ctcccccccc cctaacgtta ctggccgaag 840

ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc 900

ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg 960

tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc 1020

tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc 1080

cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa 1140

ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct 1200

ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg 1260

atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg 1320

tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg 1380

ccacaaccat ggtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc 1440

gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc 1500

cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg 1560

cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg 1620

ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga 1680

tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca 1740

cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc 1800

agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc 1860

tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt 1920

tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca 1980

ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct 2040

ccgagggccg ccaccacctg ttcctggggc atggcaccgg cagcaccggc agcggcagct 2100

ccggcaccgc ctcctccgag gacaacaaca tggccgtcat caaagagttc atgcgcttca 2160

aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg 2220

gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc 2280

ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc 2340

accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc 2400

gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg 2460

acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg 2520

taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg 2580

gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg 2640

tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg 2700

tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 2760

agcgctccga gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaagtaat 2820

ggagcaaaca tcccccaaag ttctacaaat gtggctgagg actttgtaca ttgttttgtt 2880

tttttttttt tttggttttg tcttttttta atagtcattc caagtatcca tgaaataagt 2940

ggttacagga agtccctcac cctcccaaaa gccaccccca ctcctaagag gaggatggtc 3000

gcgtccatgc cctgagtcca ccccggggaa ggtgacagca ttgcttctgt gtaaattatg 3060

tactgcaaaa atttttttaa atcttccgcc ttaatacttc atttttgttt ttaatttctg 3120

aatggcccag gtctgaggcc tccctttttt ttgtcccccc aacttgatgt atgaaggctt 3180

tggtctccct gggagggggt tgaggtgttg aggcagccag ggctggcctg tacactgact 3240

tgagaccaat aaaagtgcac accttacctt acacaaacag cttgtggctc tgtggctttg 3300

ctgggtgtgg ggagcaggtt gggtgggtgt ggagctctat tggggggggc atctagggtg 3360

ggctaggcct tgctgatggt atctagtggg agggctacag gccctttaat gaagtctcac 3420

aaataaccac accttcagac tgtcttgaag agttgaaaag gtactcctcc agaaacctag 3480

gccaagttgg cctgcactaa cactaccttc ctcaaccgag gggtgtccac accctttgcc 3540

tccatctgca taactgggtt tcaagcccct ctgctagtct gaagctgccc tttccagagc 3600

tgtgactgac agcaggaag 3619

<210> 22

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cctgaggctc ttttccagcc ttccttcttg ggtaagttgt agcctagtcc tttctccatc 60

taaaggtgac aaaactcctg aggccatagt acaagttaag tctgatttct gtcactcttc 120

tcttaggtat ggaatcctgt ggcatccatg aaactacatt caattccatc atgaagtgtg 180

acgttgacat ccgtaaagac ctctatgcca acacagtgct gtctggtggt accaccatgt 240

acccaggcat tgctgacagg atgcagaagg agattactgc tctggctcct agcaccatga 300

agatcaaggt aagctaagca tccttagctt ggtgagggtg ggccctgtgg ttgtcagagc 360

aaccttctag gtttaagggg aatcccagca cccagagagc tcaccattca ccatcttgtc 420

ttgctttctt cagatcattg ctcctcctga gcgcaagtac tctgtgtgga tcggtggctc 480

catcctggcc tcactgtcca ccttccagca gatgtggatc agcaagcagg agtacgatga 540

gtccggcccc tccatcgtgc accgcaagtg cttctaggcg gactgttact gagctgcgtt 600

ttacaccctt tctttgacaa aacctaactt gcgcagaaaa aaaaaaaata agagacaaca 660

ttggcatggc tttgtttttt taaatttttt ttaaagtttt tttttttttt tttttttttt 720

tttttaagtt tttttgtttt gttttggcgc ttttgactca ggatttaaaa actggaacgg 780

tgaaggcgac agcagttggt 800

<210> 23

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tggagcaaac atcccccaaa gttctacaaa tgtggctgag gactttgtac attgttttgt 60

tttttttttt ttttggtttt gtcttttttt aatagtcatt ccaagtatcc atgaaataag 120

tggttacagg aagtccctca ccctcccaaa agccaccccc actcctaaga ggaggatggt 180

cgcgtccatg ccctgagtcc accccgggga aggtgacagc attgcttctg tgtaaattat 240

gtactgcaaa aattttttta aatcttccgc cttaatactt catttttgtt tttaatttct 300

gaatggccca ggtctgaggc ctcccttttt tttgtccccc caacttgatg tatgaaggct 360

ttggtctccc tgggaggggg ttgaggtgtt gaggcagcca gggctggcct gtacactgac 420

ttgagaccaa taaaagtgca caccttacct tacacaaaca gcttgtggct ctgtggcttt 480

gctgggtgtg gggagcaggt tgggtgggtg tggagctcta ttgggggggg catctagggt 540

gggctaggcc ttgctgatgg tatctagtgg gagggctaca ggccctttaa tgaagtctca 600

caaataacca caccttcaga ctgtcttgaa gagttgaaaa ggtactcctc cagaaaccta 660

ggccaagttg gcctgcacta acactacctt cctcaaccga ggggtgtcca caccctttgc 720

ctccatctgc ataactgggt ttcaagcccc tctgctagtc tgaagctgcc ctttccagag 780

ctgtgactga cagcaggaag 800

<210> 24

<211> 2019

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat ggtgagcaag 600

ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac 660

ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc 720

gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc 780

cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag 840

aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt 900

ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag 960

atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg 1020

gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag 1080

gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc 1140

aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc 1200

cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg 1260

ttcctggggc atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag 1320

gacaacaaca tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc 1380

atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc 1440

cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg 1500

tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat 1560

tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 1620

ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag 1680

gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg 1740

ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc 1800

caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac 1860

atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc 1920

acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac 1980

cacctgttcc tgtacggcat ggacgagctg tacaagtaa 2019

<210> 25

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cttcaaggtg aaatgcttga ctcctagact tgtgacccag caaagtgctc tataggtagg 60

gttactaggt cagagagtct tgcctgcaaa ccacaattat acaattccac caaatgtaag 120

acatgtaaaa atgacttatt tagctcaggt tttgaaaatt aaaaatcaaa cccacaaaga 180

cctaactttc acattaagta caaatgttta acatatataa catgttttgc cttgatatat 240

gaaatcatat gacatcacct gacaggaaac ttaagtttat ttgaacaaca atcagcctaa 300

ggtaggccta gcacatgatc aggatagtgc agggaaaccc aaagaagtgc ttctgagtat 360

aaaccaagag acctcgaaat agcagctttg ttctgtatct catgggcttt aactatcact 420

tgaaaacaat ttcacaaatc acaaaaccaa catttgttta gtttccaact ctgcctattc 480

accggagaaa tccatgtccc aatttgatgg gggaaaactt gaatgaaata ttatttggaa 540

tattttaata tccctgtgta tatgcagatg gtttaaagac aggtaacctt aactgtagtt 600

tcttggttgt ctgccttgtt ttttgaaata tgctctcacc aggagcctgc caagtaacta 660

ctcttgtgtg ctcagcaagt cctagggatc ccttaagcat gctctaacag gcctggcttt 720

tttttttaaa aagtaggttg cggtgataat aattcaagtt ttcatcactt gaaacacatt 780

ttaccaacta tcacccaagc 800

<210> 26

<211> 799

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tcccttagtg tgtcccctat aaaagaatct gacctgcaag ttccaaaagt attactacat 60

tttaacctta gtattcagga gacaaaagca ggcagaactt tagtctacat atcttgttgc 120

agatcaggca gagatacata gtgagaccct gcctcaggaa aagctatcat ttttatattc 180

acagtaagca aattaacatt aaaagtcaga aattcttaat ttgactttgg ctgtgaagaa 240

tttggattct ctggatttga aactagaaaa tctgattaag cgatgcaaca gtttctgtat 300

actaaaactc taagaccctt ggttctaaag ataccacatt taaaaaaaaa gttaagtata 360

agacaaaaag ccttaatagc tcttttatag gggagggact catttaatat tagtccacct 420

cactcctcat aactatttta tgaggtttat tatataaccc attttttaaa aaaaaaatta 480

aaggacaatt ttagtgtttg aaagatttcc caaccccacc ctggaaatca ggctgcaaat 540

ctcagcagct gcccttagaa ggcaaaaagc catgccattt aagccatggg aagttagtag 600

caaacaagag accacatggc aggaaggagg ctttaaagaa agcccacagt gtcaaaagac 660

ccaaccaaca gcagagacag aaaaaataat acccaaatct catcagaaag gtagacggat 720

ttagccacat ccatagtggc tcattaggga atgcttcaat tataatttta tgaaatctca 780

gagcccccct ccccaacag 799

<210> 27

<211> 1592

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ctcgaggcca ccatggccca gtccaagcac ggcctgacca aggagatgac catgaagtac 60

cgcatggagg gctgcgtgga cggccacaag ttcgtgatca ccggcgaggg catcggctac 120

cccttcaagg gcaagcaggc catcaacctg tgcgtggtgg agggcggccc cttgcccttc 180

gccgaggaca tcttgtccgc cgccttcatg tacggcaacc gcgtgttcac cgagtacccc 240

caggacatcg tcgactactt caagaactcc tgccccgccg gctacacctg ggaccgctcc 300

ttcctgttcg aggacggcgc cgtgtgcatc tgcaacgccg acatcaccgt gagcgtggag 360

gagaactgca tgtaccacga gtccaagttc tacggcgtga acttccccgc cgacggcccc 420

gtgatgaaga agatgaccga caactgggag ccctcctgcg agaagatcat ccccgtgccc 480

aagcagggca tcttgaaggg cgacgtgagc atgtacctgc tgctgaagga cggtggccgc 540

ttgcgctgcc agttcgacac cgtgtacaag gccaagtccg tgccccgcaa gatgcccgac 600

tggcacttca tccagcacaa gctgacccgc gaggaccgca gcgacgccaa gaaccagaag 660

tggcacctga ccgagcacgc catcgcctcc ggctccgcct tgccctgact cgaggggcca 720

ccgtatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg 780

tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 840

tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 900

gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 960

tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 1020

tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 1080

ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc 1140

gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg 1200

ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 1260

gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 1320

ctccccgcat cgataccgtc gatcctgtgc cttctagttg ccagccatct gttgtttgcc 1380

cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 1440

atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 1500

ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 1560

gctctatggg atgagggcaa tctgggaagg tt 1592

<210> 28

<211> 1753

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gcaacactcc cctcccccac ataacttacg gtaaatggcc cgcctggctg accgcccaac 60

gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 120

ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 180

gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 240

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 300

gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc 360

cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg 420

ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc 480

ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct 540

tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgggg 600

agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 660

cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 720

cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 780

gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 840

tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 900

gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 960

gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt 1020

gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc 1080

cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 1140

gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 1200

ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg 1260

gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 1320

agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 1380

cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 1440

gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 1500

ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 1560

ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc 1620

tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg atttgatacc 1680

gcgggcccta aacgcgtacc aacgtgaaaa aattattagg taccttcgcg gccggccttc 1740

acgagccgcc acc 1753

<210> 29

<211> 4760

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 60

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 120

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 180

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 240

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 300

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 360

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 420

agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 540

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 600

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 660

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 720

accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 840

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 900

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 960

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1020

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1080

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1140

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1200

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1260

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1320

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1380

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1440

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1500

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1560

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1620

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1680

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1740

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1800

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1860

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1920

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1980

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2040

tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 2100

atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga 2160

cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 2220

agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 2280

aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg 2340

tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt 2400

ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg 2460

taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg 2520

atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg 2580

gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt 2640

aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac 2700

ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag 2760

accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc 2820

tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc 2880

ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg 2940

cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt 3000

tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg 3060

ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac 3120

gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc 3180

atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccagtg atcgaagtta 3240

ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc 3300

tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa 3360

tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg 3420

ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga 3480

cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg 3540

tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc 3600

ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg 3660

cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga ggaaactcgg 3720

ccgtggtgac caatacaaaa caaaagcgct cctcgtacca gcgaagaagg ggcagagatg 3780

ccgtagtcag gtttagttcg tccggcggcg ccagaaatcc gcgcggtggt ttttgggggt 3840

cgggggtgtt tggcagccac agacgcccgg tgttcgtgtc gcgccagtac atgcggtcca 3900

tgcccaggcc atccaaaaac catgggtctg tctgctcagt ccagtcgtgg actgacccca 3960

cgcaacgccc aaaataataa cccccacgaa ccataaacca ttccccatgg gggaccccgt 4020

ccctaaccca cggggcccgt ggctatggca gggcctgccg ccccgacgtt ggctgcgagc 4080

cctgggcctt cacccgaact tggggggtgg ggtggggaaa aggaagaaac gcgggcgtat 4140

tggccccaat ggggtctcgg tggggtatcg acagagtgcc agccctggga ccgaaccccg 4200

cgtttatgtg gacctgggac cgcgccgccc cgactgcatc tgcgtgttcg aattcgccaa 4260

tgacaagacg ctgggcgggg tttgctcgac attgggtgga aacattccag gcctgggtgg 4320

agaggctttt tgcttcctct tgcaaaacca cactgctcga cattgggtgg aaacattcca 4380

ggcctgggtg gagaggcttt ttgcttcctc ttgaaaacca cgagtttacc actccctatc 4440

agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga 4500

aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcact gctcgactct 4560

agaggatcgc ggccggccgc agcttgtcga ccgcggcctt tagcgagttt accactccct 4620

atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag 4680

tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc ttatcgatga 4740

taagctgtca aacatgagaa 4760

<210> 30

<211> 100

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ggggacacac uaagggagcu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tgtgttgtgt gtgcttgtgc 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

caatccagcg gaccttcctt 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

agagtgaagc agaacgtggg 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

agacctgttg ctgctcagac 20

<210> 35

<211> 1887

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ttaggtacct tcgcataact tcgtatagca tacattatac gaagttatcc tcagcaccat 60

ggctagcggc agcctcggag tttgaataga tagaataaaa tatctttatt ttcattccat 120

ctgtgtgttg gttttttgtg tgagatctac gggtggcatc cctgtgaccc ctccccagtg 180

cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta 240

agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg 300

gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa 360

ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa 420

gcgattctcc tgcctcaggc tcccgagttg ttgggattcc aggcatgcat gaccaggctc 480

agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc 540

aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg 600

tgaaccactg ctcccttccc tgtccttctg cctcagctaa ttgagtaggg gggaggctaa 660

ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac 720

agaataaaac gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc 780

tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc 840

ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg 900

gcggcaggcc gtggaattcg taaatgaatt ttctgtatga ggtcgcgatg aataaatgaa 960

agcttgcaga tctgcgactc tagaggatct gcgactctag aggatcataa tcagccatac 1020

cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 1080

acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 1140

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 1200

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgcgact ctagaggatc 1260

ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc 1320

cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct 1380

tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 1440

ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctgc 1500

gactctagag gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa 1560

acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 1620

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 1680

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 1740

atgtctggat ccccatcaag ctgatccgga acccttaata taacttcgta tagcatacat 1800

tatacgaagt tattaggtcc ctcgacctgc agcccaagct agatcgaatt cggccggcct 1860

tcacgagccg ccaccctcga ggccacc 1887

<210> 36

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

acatacagct gtacataaag taactatcag ttaggcttgt gtcaactgtt tggatttttt 60

tcacttgaat atttgggact ttttcttttg gtttattaaa agttacatat gccacgtgtg 120

tgaacgatat ggctggtact gtgtttattt cttccatgaa ctaagacagt ctaaatgagt 180

tcctttcacg ttttaatttt accttaggac ttctggaatt tcttctgcac ataaagttct 240

gatagcatta gtttaagctg gactaaccct gaaagtagct tgtggcaagt atcaaggaat 300

caatattata ctctacaaaa tcaaagttta cagagaagtc atatagtaat ttttctgaaa 360

tttactggca caatgttaat ccagcctgac tccaactaat taatggtcac attaatttaa 420

gtctttccct tgcctctgct gcattagttt ctctcaaaat tgttaactta caacttgaag 480

tctggtatta taaattgaat gtaaagcatt ctgaaagata ctatactgat tgcaggtttt 540

tcagtcaggt tcaagctaat ttgaccagtc attggattaa ttatggatct ggggccataa 600

atgctatttt aattccacta tagagattaa aataagccat tctccatttc ataatattct 660

attggacttt gactgcaggg gcctccaagt cttgacagta gattataatc cttcagctgc 720

ccactctact ggaggaggac aaactggtca cttttcagca aaacctggct gtggatcagg 780

gcagtctggt acttccaagc 800

<210> 37

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tcattagatg ccatcatgct ctcactgcct cctcagcttc aagaggaatc tggaaaaagc 60

agtcccactg gtcaggaaag gaacactagt gcacttatcc tgggtgtctg ctgagccaac 120

agtggtagta aggtaagggc aggatgtgtc aaactgccaa tagagaacta cttactcttc 180

aggctgaagc tgatggaaca ggtaacaaag gcaaacacta atcatgatca gcaagatgaa 240

gcagaaaggg aacaagggga tattaaatgt gtatagacac gctagagaga tggctcagca 300

gttaagagaa ctagctggtc tttcagaggt cctgagatca attttagaca cccacatggt 360

ggctcatgac catctatcta taaatggatc tgattttcat gtctggcagt gtacagaagc 420

taactgaaga aaggtggaag acccacaaga gttcaagata agccctatat agtgaagttc 480

aaggcaagct ttttctacct gaaacttagt ctcaaaaaaa aatgaatacg taaacagtct 540

tccaggggat aagaacctta cagaaaaagc agaaatgcct ggggcactgg attaccgatg 600

taatcaaatt cagtccttga attgaacaca ggattgccta gagcaaggcc agccagagat 660

tcatctcaga gggagaaagg tgtctttgga gcaattttgt ggtaatctag tatgtatcac 720

ataagtttag acgcatttgg gactggaaag atgtgaacaa agcaccctat ggctcacatc 780

tgtcattaac tctagttcca 800

<210> 38

<211> 768

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac 60

aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc 120

atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact 180

agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc 240

aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc 300

gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag 360

atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg 420

gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg 480

gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc 540

aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa 600

agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga 660

tactgcgacc tccctagcaa actggggcac aagcttaatg actacaaaga ccatgacggt 720

gattataaag atcatgatat cgattacaag gatgacgatg acaagtag 768

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

tgcatcctcc aggctctagt 20

<210> 40

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

agagtgaagc agaacgtggg 20

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

caatccagcg gaccttcctt 20

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

cacaacctgg tcagtccctc 20

<210> 43

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gggaccgcag cactggggag gcggtccgct cccagaatgg taagggcgag agaatggtaa 60

gggtccagct cgcggcggcg caggccgaag cccagccgca gtccgtaggc cggggctgtc 120

cgccgggccc agtgaagttc cttcaggaga agcccagcca tcccggatcc gccctcacta 180

gcggccggct cggcgccatt gcgccctacc gtgtaaggga agaatgttcg agaatgcaca 240

cggttggggg aggcgttcaa gatgggtgtg ggcgtccgtg ccccaatacc tagtagagga 300

gctccagata gacttctccg ccactcccct ctgctccttt gttgggttcc aagccggttg 360

gagcacctct gacatagcca gaaaagagta gctttcaccg gttccattgg cccttgagaa 420

gatgctcata gaatctaggt ggcttttgcg ttgcttccct tccctggtct ggttcttgga 480

tgcccagaat ctgcaggtga cagctgctcc cccagtaaaa cgtaacggaa tgcaaactga 540

acgttattaa gcgatacttt tagctgcctt tatcgtggtt aactttaata cgagtaagca 600

cgtggtttgc tgtggttgaa tttgttcttg aaagcattcc cttgcaggaa ccgtgttctg 660

tgttgtgctt tttttttttt tttaaacaac tctcttagaa attactacta ctttatcctg 720

gtgaactatc taatattctt actaaacttg ccagagtctg aagagaaagg ctgaacttaa 780

agggtttttt cccccctagt 800

<210> 44

<211> 1169

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

caacctggcc gttaagatgc ataacttcgt ataatgtatg ctatacgaag ttatgtagta 60

gtaaggaatg aacttaagat tgcttataat ttgaggcatt aaaatctttg caatcggtga 120

tataccaata ctaaatagaa aaaagaatag ttaaccttca ctggtcctta agtaagtgat 180

tccccccccc ccccacacac acacacacac acacacacag ggtttcactg tatagctgtg 240

gctgtcctgg aagcaacttt gtagaccagg ctggcctcga actcagaaat ctgcctgcct 300

ctgcctcccg agtgctggga ttaaaggcgt gcgccgccac acccggcttg attttttttt 360

ttttaaatag taattacaga ctttagattt tagcaaatca taggtagttg agtgtcatag 420

tttatgtgtt gttctaatta gttttcactg ttcatctaca gttatggctg cagcttctat 480

tgccaacatt gttaaaagtt cttttgggcc agttggcttg gataaaatgt tggtggatga 540

tattggtgta agtatatatc taattgctct tgcttgcctt gataaaagta cattgtttta 600

aatttcaaaa aaaaatcttt ggtaaagtct ttgtttttac ctgagtatgg atggcatcct 660

agagcttggt ttctgtgttg accacttatt ccttcatatt tcttaggatg taaccattac 720

taacgatggt gccaccatcc tgaagttact ggaggtagaa catcccgcag ccaaagttct 780

gtgtgagctg gctgacctgc aagacaaaga agttggagat ggaactacct cagtggtgag 840

acgctaccag gaaatggcta gagaatagtc aatttattag agttgaatcc atgagagagt 900

catactctag gtcagaaata agtcaccttt taaggcagtc tcacaaactt ttgaaaacta 960

accattgtaa ctagtttgtg taatttaagt aagggaagaa cttaaggtga ttaaataaat 1020

acaagcttgt tttaaatact ggagtctcgt gaatttggag ccttatttaa acctacttaa 1080

ccatgatgat gttcatgtag aaaatgttac cagataactt cgtataatgt atgctatacg 1140

aagttattaa ccactggtgg cattgatga 1169

<210> 45

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gtttggactt ttttctttgt tgtttaatag atacttttgt attttaaatg tgaacaagaa 60

atctttggaa ttcaaaatag aaatttgcag aagttaattt gctaccatat agtgctaatg 120

gctagtcagc aaacatggcg aatagtgtaa cattttgtga gttagcctgc ttaagtagta 180

aaatactgtt tggtgaggca gattttagac ctggcaaaat aaatggtgag ggaaaagtat 240

ttatagactt accctttact aggaaagaat tgaatctgtt tttaaatagt gaaaatggac 300

tttatctaat ttgttcatag acaatgtaac ttaaaaataa ccttaattcc actcgaaaga 360

agaaaagttt tttggagatt ttgttttgtt ttattattaa gaaaactggt ttttcacagt 420

gttctctatt ttaaatgatc ttaagatttt attgaaatta ctggaatgat accagtcatt 480

tccattgatt gctatctagc tcttgttgca gtaagtctgt aaatgtaatg gatataaatt 540

ctgctgtatt agatttttag cattttctgg cttttccagc ttggaccatt tccatagaat 600

acttttaggt tttttgaggc aggatttctc tgtgtagccc tgactgtcct ggagctcact 660

ttgtagacca ggctggcctc gaactcagaa atccgcctgc ctctacctcc caagtgctag 720

gattaaaggc atgcgccacc acgcctggca cagaatactt cctagtggtc tctatgttgt 780

gttttcaacg agttgctact 800

<210> 46

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

atccctaccg ctttcccggc a 21

<210> 47

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

tcatcaatgc caccagtggt ta 22

<210> 48

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

caacctggcc gttaagatgc 20

<210> 49

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cgatagccac taataactga t 21

<210> 50

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

ggcctgtgcc tggggtggcc tactctgtgt cactcccggc ctccttcctg gaggatgtag 60

ccggcagcgg ggaagctgag ggttcttcag cctcttcccc gagcctgccg ccgcctggga 120

ctccagcctt cagtcccaca ccggagagac cccagcccac agctctggac ggccccgtgc 180

cacccaccaa cctcctggaa gggatcatgg atttcttccg gcagtacgtg atgctcatcg 240

cggtggtggg ctcgctgacc ttcctcatca tgttcatagt ctgcgccgcc ctcatcacgc 300

gccagaagca caaggccaca gcctactacc catcctcgtt ccctgaaaag aagtatgtgg 360

accagagaga ccgggctggg ggaccccgta ccttcagcga ggtccctgac agggcacctg 420

acagccggca tgaagaaggc ctggacacct cccatcagct ccaggctgac attctggctg 480

ctacccagaa cctccggtct ccagctagag ccctgccagg caatggggag ggagcaaagc 540

ctgtgaaggg tgggtcggag gaggaggagg aagaggtgct cagcggtcag gaggaggccc 600

aggaagcccc agtatgtggg gtcactgaag agaagctggg ggtcccagag gagtcggtct 660

cagcagaggc tgaaggggtt cctgccacca gtgagggcca aggggaagca gaagggtctt 720

tctccttagc ccaggaatcc cagggagcaa ctggtcctcc tgaaagtccc tgtgcctgca 780

acagagtctc ccccagtgtc 800

<210> 51

<211> 1563

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

gactacaaag accatgacgg tgattataaa gatcatgata tcgattacaa ggatgacgat 60

gacaagggaa gcggagctac taacttcagc ctgctgaagc aggctggcga cgtggaggag 120

aaccctggtc ctatggtgag caagggcgag gaggtcatca aagagttcat gcgcttcaag 180

gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc 240

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc 300

ttcgcctggg acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac 360

cccgccgaca tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc 420

gtgatgaact tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac 480

ggcacgctga tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta 540

atgcagaaga agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc 600

gtgctgaagg gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg 660

gagttcaaga ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg 720

gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag 780

cgctccgagg gccgccacca cctgttcctg gggcatggca ccggcagcac cggcagcggc 840

agctccggca ccgcctcctc cgaggacaac aacatggccg tcatcaaaga gttcatgcgc 900

ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc 960

gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc 1020

ctgcccttcg cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg 1080

aagcaccccg ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg 1140

gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg 1200

caggacggca cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc 1260

cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc 1320

gacggcgtgc tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac 1380

ctggtggagt tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac 1440

tacgtggaca ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag 1500

tacgagcgct ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag 1560

taa 1563

<210> 52

<211> 800

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

ggctctgggt tagctgtctc aaaggttgct aggtccgtgg caggccccag aactgctggg 60

acccgaatgt tgggtccttg agggtcacct ctttggtcaa gaaaggcatt cagctctaac 120

tgctccttga taccacgtgg cttggccatt gctggtgcca aggctgaccc cgaactggca 180

gagccgatgc cctctggtgc accccaggaa acatctcccc aagttccagc gcccttaatg 240

actcttgcca ccctgggggc ttcaccctaa cgcaccactt ctctggaagg ggaaggccag 300

acacatgcca gttggggctg catgaggcag tcctcagagc agaaggggac caggccagag 360

gccacctgtg acggggcaaa ctgcatctcg gctgtggaga ccagaggggc tgttagattt 420

ggaagacatc aatgactggg cctgcggcgc agcccgtgtc tggtaatacc agggacggca 480

gaggcgtttg catcttccca tcacctgcaa tgtcgctgtc actctgcccc tgttcagtgg 540

acttgattag ttaggaaact tctggaaggg gccccctact ttatatcaca gagtttgccc 600

tagacacccc gtggaaacac aaactcaaaa tcaagtggct ttaggagccg ctgtgcccct 660

ccacaagctg acatggctac tctaagggtt cctgctgggc tggctttgct acgctttcct 720

caagctgctt tcttattacc aggatgcctc acagctacaa agtccaatct cacagcaccc 780

gcactggaaa atactgttct 800

<210> 53

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

ataacctatc ccctaacaca taagcaccag 30

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

cagcttggtg tccacgtagt 20

<210> 55

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

catggacgag ctgtacaagt aa 22

<210> 56

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

tggaatggta tttgggtgtt 20

Claims (17)

1. A fusion protein comprises a Cas9 protein and a TetR protein, wherein the Cas9 protein and the TetR protein are connected through a flexible connecting peptide.

2. The fusion protein of claim 1, wherein the flexible linker peptide consists of 1-6 GGGGS domains.

3. The fusion protein of claim 2, wherein the flexible linker peptide consists of 4 GGGGS domains.

4. The fusion protein of any one of claims 1-3, wherein the coding sequence of the Cas9 protein is shown as SEQ ID NO 1, and the coding sequence of the TetR protein is shown as SEQ ID NO 2.

5. The fusion protein of claim 4, wherein the encoding nucleic acid sequence of the fusion protein is shown as SEQ ID NO. 13, and the protein sequence thereof is shown as SEQ ID NO. 14.

6. A homologous recombination system comprising a Cas9-TetR fusion protein, a gRNA, a vector containing a tetracycline operator (tetO) element.

7. The homologous recombination system of claim 6, wherein the tetO sites are located outside the left arm and/or the right arm of the homologous recombination of the recombination vector, and the number of tetOs is 1-7 per arm.

8. The homologous recombination system of claim 7, wherein 3 tetO sites are added simultaneously outside the left arm of homologous recombination and outside the right arm of homologous recombination.

9. The homologous recombination system of claim 8, wherein the vector containing the tetracycline operator (tetO) element is a DNA vector.

10. The homologous recombination system of claim 9, wherein the vector comprises a left arm of homologous recombination, an insertion element, and a right arm of homologous recombination, all connected in sequence from 5 'to 3'.

11. The homologous recombination system of claim 10, comprising a gRNA expression vector or a Cas9-TetR expression vector or a Cas9-TetR-gRNA expression vector.

12. Use of the fusion protein of any one of claims 1 to 5 or the homologous recombination system of any one of claims 6 to 11 for increasing the positive rate of homologous recombination.

13. The use according to claim 12, wherein the vector for homologous recombination is a DNA fragment of more than 2000 bp.

14. Use of the fusion protein of any one of claims 1-5, the homologous recombination system of any one of claims 6-11, for the preparation of a genetically modified animal model.

15. A method of gene recombination comprising:

step 1: gene synthesis or plasmid template amplification mode to obtain homologous recombination left arm, homologous recombination right arm, and inserted element sequence segment, PCR amplification, and connecting the vector and the segment to form homologous recombination vector;

step 2: designing a gRNA sequence aiming at a genome site of a target cell, and constructing a Cas9-tetR expression vector and a gRNA expression vector; or in vitro transcription to obtain Cas9-tetR mRNA, and artificially synthesizing gRNA;

and step 3: introducing the homologous recombination vector obtained in the step 1, the Cas9-tetR and gRNA expression vector obtained in the step 2, or the Cas9-tetR mRNA and the artificially synthesized gRNA into a target cell together.

16. The gene recombination method according to claim 15, wherein the vector in step 1 is a plasmid vector comprising tetO.

17. The method of gene recombination according to claim 15, wherein the introduction in step 3 is microinjection or cotransfection.

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