CN114958758B

CN114958758B - Construction method and application of breast cancer model pig

Info

Publication number: CN114958758B
Application number: CN202110187956.7A
Authority: CN
Inventors: 牛冬; 汪滔; 陶裴裴; 曾为俊; 王磊; 程锐; 黄彩云; 赵泽英; 马翔; 段星; 刘璐
Original assignee: Nanjing Qizhen Genetic Engineering Co Ltd
Current assignee: Nanjing Qizhen Genetic Engineering Co Ltd
Priority date: 2021-02-18
Filing date: 2021-02-18
Publication date: 2024-04-23
Anticipated expiration: 2041-02-18
Also published as: CN114958758A

Abstract

The application provides a pig cell expressing PyMT, a breast cancer model pig obtained from the pig cell through a somatic cell cloning technology, a construction method of the pig cell and application of the pig cell in the field of biological medicine. Comprising inserting a nucleotide sequence encoding PyMT into a safe harbor site of a pig to obtain a polypeptide expressing SEQ ID NO:14 and a breast cancer model pig, wherein the pig safe harbor site is selected from the group consisting of pig ROSA26, AAVS1, H11 and COL1A1 safe harbor site. The application has good applicability of the research object, high expression level of target genes in pig cells and high gene editing efficiency.

Description

Construction method and application of breast cancer model pig

Technical Field

The invention relates to the technical field of gene editing, in particular to a pig recombinant cell which is integrated at a specific position in a genome and is driven to express a PyMT oncogene by a mammary gland specific promoter MMTV-LTR and is constructed by a CRISPR/Cas9 system and a homologous recombination technology, wherein the recombinant pig cell is used for cloning and producing a breast cancer model pig, and the model pig can be used in the biomedical fields of drug screening and drug effect evaluation in the next step, gene and cell therapy, study of a breast cancer pathogenesis and the like.

Background

According to the latest global cancer burden data issued by the international cancer research Institute (IARC) of the world health organization in 2020, 60% of new cancer cases in 2020 come from the most common 10 kinds of tumors, wherein female breast cancer accounts for 11.7% of the most common tumors, and the number of female breast cancer exceeds lung cancer (11.4%) for the first time, so that the cancer is the most diagnosed cancer worldwide. Female breast cancer is the first cancer to be diagnosed most often, is the first killer to threaten female health, and is one of the most urgent health problems today. With the continued development of modern medicine, despite advances in cancer diagnosis, treatment, and longevity, mortality has not been improved to a great extent. The lack of understanding of the natural history of the disease is the main reason for this limitation, and it is currently unclear at the molecular level which changes in breast tumors may lead to invasion and metastasis.

MMTV is an important virus that causes mouse mammary tumors, and its viral tissue specific promoters such as the mouse mammary tumor virus long terminal repeat promoter (MMTV-LTR) can be used to mediate the high expression of oncogenes in the mouse mammary glands to develop breast cancer. The polyoma intermediate T antigen (polyoma MIDDLE T ANTIGEN, pyMT, encoded amino acid sequence shown as SEQ ID NO: 14) is an annexin encoded by small DNA polyoma virus. PyMT was found to induce tumor production, and its induced tumors were also prone to tissue metastasis, and biomarker expression was also consistent with that associated with a poor prognosis in humans. PyMT acts as a powerful oncogene, whose product binds and absorbs several signal transduction pathways, including the Src family, ras and PI3k kinase pathways, all of which are altered in human breast cancer.

The mechanism of tumor development and treatment research are all required to be carried out on the basis of corresponding animal models, and the animal models which are commonly used at present are mouse models, for example, patent TW201536324A discloses a method for increasing the effectiveness of medical therapy, wherein in the embodiment, the injection of MMTV-PyMT cells into p16-3MR transgenic mice is generally mentioned, and then treatment and the like are carried out to obtain breast cancer cell models. However, mice differ greatly from humans in terms of body size, organ size, physiology, pathology, etc., and cannot truly simulate normal physiological and pathological conditions of humans. Furthermore, the mouse breast cancer model obtained by injecting MMTV-PyMT cells is also not stably inherited. Furthermore, patent CN103173496B discloses a tree shrew breast cancer model, which is established by injecting lentivirus into the nipple, specifically, firstly constructing a viral vector, then packaging the virus and determining the titer, and finally injecting lentivirus into the nipple of the tree shrew. However, the tree shrew is not a common disease animal model, the size and physiological function of the tree shrew are not high in similarity with those of a human, and normal physiological and pathological states of the human cannot be truly simulated. Thus, there remains a need for more and more suitable animal models of breast cancer, including further improvements in animal selection and in methods of making animal models. Pigs are major meat animals for a long time, have the size and physiological functions similar to those of human beings, are easy to breed and raise on a large scale, have lower requirements on ethical morals, animal protection and the like, and are ideal human disease model animals.

Therefore, the application adopts the gene editing technology and the mammary gland specific promoter MMTV-LTR to construct the pig recombinant cell for the mammary gland tissue specific expression oncogene PyMT, and then uses the recombinant cell as a nuclear transfer cell donor clone to produce the breast cancer model pig, and the model pig obtained by the application becomes a powerful tool for researching the generation mechanism of breast cancer diseases, drug development and preclinical test.

Disclosure of Invention

The invention provides a method for inserting a nucleotide sequence for coding PyMT into a safe harbor site of a pig at fixed points by a gene editing method to prepare a pig cell for expressing the PyMT protein, and the pig cell can further produce a breast cancer model pig for expressing the PyMT protein in breast tissues by a somatic cell cloning technology, so that a powerful experimental tool is provided for pathogenesis of diseases and research and development of therapeutic drugs.

In a first aspect of the present invention, there is provided a pig cell expressing PyMT, the nucleotide sequence encoding PyMT being inserted into a safe harbor site of a pig to obtain a polypeptide expressing SEQ ID NO:14, porcine cells of PyMT.

Preferably, the inserted nucleotide sequence encoding PyMT may be the CDS sequence or cDNA sequence of PyMT.

Preferably, the amino acid sequence of the PyMT is SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In one embodiment of the present invention, the inserted nucleotide sequence encoding PyMT is as set forth in SEQ ID NO: 39.

Preferably, the swine safe harbor site is selected from swine ROSA26, AAVS1, H11 or COL1A1 safe harbor site.

In one specific embodiment of the invention, the nucleotide sequence of each 500bp of the ROSA26 safe harbor site region and the upstream and downstream thereof is shown in SEQ ID NO:40, and the nucleotide sequence of 500bp respectively at the AAVS1 safe harbor site region and the upstream and downstream thereof is shown as SEQ ID NO:41, and the nucleotide sequence of 500bp respectively at the upper and lower reaches of the H11 safe harbor site region is shown as SEQ ID NO:42, the nucleotide sequence of 500bp of each of the COL1A1 safe harbor site region and the upstream and downstream thereof is shown as SEQ ID NO: 43.

Further preferably, the optimal safe harbor site of the pig is a COL1A1 site.

Preferably, the nucleotide sequence encoding the PyMT is regulated in pig cells by an exogenous promoter, and the exogenous promoter is MMTV-LTR.

Mouse Mammary Tumor Virus (MMTV) Long Terminal Repeat (LTR) -driven transgenes allow targeted expression of various oncogenes and growth factors in mammary tumor transformation.

In one embodiment of the invention, the nucleotide sequence encoding PyMT is driven in porcine cells by MMTV-LTR, the nucleotide sequence of MMTV-LTR is as set forth in SEQ ID NO: 15.

Preferably, the porcine cells are somatic cells of a pig. Further preferred are somatic cells of any pig that can be used in somatic cell nuclear transfer technology.

Preferably, the porcine cells may be breast cells, embryonic stem cells, adult stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, hepatic stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, somatic cells, fibroblasts, muscle cells, glial cells, adipocytes, germ cells, or the like.

In one embodiment of the invention, the porcine cell is a porcine fibroblast or a mammary gland cell (preferably a mammary gland epithelial cell).

In a second aspect of the present invention, a construction method of the pig cell is provided, wherein a nucleotide sequence encoding the PyMT is inserted into a safe harbor site of the pig to obtain a nucleotide sequence expressing SEQ ID NO:14, porcine cells of PyMT.

Specifically, gene editing based on homologous recombination, ZFN, TALEN, CRISPR/Cas9 editing technology based on nuclease and the like can be adopted.

Preferably, the construction method comprises inserting a nucleotide sequence encoding a PyMT into a pig safe harbor site using a safe harbor site vector comprising a nucleotide sequence encoding a PyMT and a safe harbor site vector backbone comprising a 5 'homology arm and a 3' homology arm of the safe harbor insertion site, wherein the nucleotide sequence encoding a PyMT is located between the 5 'homology arm and the 3' homology arm, and the safe harbor site vector backbone is selected from any one of the following:

A) The ROSA26 safe harbor site vector skeleton, the 5' homology arm of which is shown in SEQ ID NO:5, the 3' homology arm is shown in SEQ ID NO: shown at 6. Preferably, the nucleotide sequence of the ROSA26 safe harbor site vector skeleton is shown in SEQ ID NO: 4.

B) AAVS1 safety harbor site carrier skeleton, its 5' homology arm is as SEQ ID NO:7, the 3' homology arm is shown as SEQ ID NO: shown at 8. Preferably, the AAVS1 safe harbor site vector backbone has a nucleotide sequence obtained by combining the nucleotide sequence of SEQ ID NO:4, the 5 'and 3' homology arms of ROSA26 are replaced with the 5 'and 3' homology arms of AAVS 1.

C) H11 safe harbor site carrier skeleton, the 5' homology arm of which is shown in SEQ ID NO:9, the 3' homology arm is shown as SEQ ID NO: shown at 10. Preferably, the nucleotide sequence of the H11 safe harbor site vector skeleton is a nucleotide sequence obtained by combining SEQ ID NO:4, the 5 'and 3' homology arms of ROSA26 are replaced with the 5 'and 3' homology arms of H11.

Or D) a COL1A1 safe harbor site carrier skeleton, wherein the 5' -homology arm is shown in SEQ ID NO:11, the 3' homology arm is shown as SEQ ID NO: shown at 12. Preferably, the nucleotide sequence of the COL1A1 safe harbor site vector skeleton is a sequence obtained by using SEQ ID NO:4, the 5 'and 3' homology arms of ROSA26 are replaced with the 5 'and 3' homology arms of COL1 A1.

Further preferably, the pig optimal safe harbor site vector skeleton is a COL1A1 safe harbor site vector skeleton.

Preferably, the safe harbor site vector further comprises a promoter, a signal molecule and nucleotide sequences encoding EGFP protein, mCherry protein and puro resistance protein. Wherein the promoter is EF-1 alpha promoter, PGK promoter and/or pCAG promoter. The signal molecules are EF-1 alpha poly (A) signal, bGH poly (A) signal and/or beta-globin poly (A) signal. Further preferably, the insulating region is also included.

In one embodiment of the present invention, the safe harbor site vector backbone comprises, in order from 5 'to 3', a 5 'homology arm, an insulator region, an EF-1. Alpha. Poly (A) signal, a nucleotide sequence encoding EGFP, an EF-1. Alpha. Promoter, an insulator region, a PGK promoter, a nucleotide sequence encoding mCherry, a bGH poly (A) signal, a loxP-puro-loxP expression cassette region, an insulator region, a beta-globin poly (A) signal, a pCAG promoter, an insulator region, and a 3' homology arm.

In one specific embodiment of the invention, the nucleotide sequence of the COL1A1 safe harbor site vector is shown in SEQ ID NO: shown at 13.

Preferably, construction of porcine cells is performed using an sgRNA vector comprising a sgRNA targeting the ROSA26, AAVS1, H11 or COL1A1 safe harbor site, wherein:

The nucleotide sequence of the sgRNA targeting the ROSA26 is shown in SEQ ID NO:21, the nucleotide sequence of the sgRNA targeting AAVS1 is set forth in SEQ ID NO:22, the nucleotide sequence of the sgRNA targeting H11 is set forth in SEQ ID NO:23, the nucleotide sequence of the sgRNA targeting COL1A1 is shown in SEQ ID NO: shown at 24.

Preferably, the sgRNA vector further comprises a backbone vector, and the nucleotide sequence of the backbone vector is SEQ ID NO:3.

Preferably, the construction of the pig cell is performed using a Cas vector comprising nucleotide sequences encoding Cas proteins, EGFP and Puro resistance proteins, wherein the Cas vector further comprises an EF1a promoter, a WPRE element and a 3' ltr sequence element, preferably, the nucleotide sequence of the Cas vector is, in order from 5' -3 ': a CMV enhancer, an EF1a promoter, a nuclear localization signal, a nucleotide sequence encoding a Cas protein selected from Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas5d、Cas5t、Cas5h、Cas5a、Cas6、Cas7、Cas8、Cas9、CaslO、Csyl、Csy2、Csy3、Csy4、Csel、Cse2、Cse3、Cse4、Cse5e、Cscl、Csc2、Csa5、Csnl、Csn2、Csml、Csm2、Csm3、Csm4、Csm5、Csm6、Cmrl、Cmr3、Cmr4、Cmr5、Cmr6、Csbl、Csb2、Csb3、Csx17、Csx14、CsxlO、Csx16、CsaX、Csx3、Csxl、CsxlS、Csfl、Csf2、CsO、Csf4、Csdl、Csd2、Cstl、Cst2、Cshl、Csh2、Csal、Csa2、Csa3、Csa4、Csa5、C2cl、C2c2、C2c3、Cpfl、CARF、DinG、 a homologs or modified forms thereof, preferably Cas9, a nuclear localization signal, a nucleotide sequence encoding a cleavage polypeptide P2A, a nucleotide sequence encoding an EGFP, a nucleotide sequence encoding a cleavage polypeptide T2A, a nucleotide sequence encoding a Puro resistance protein, a WPRE sequence element, a 3' ltr sequence element, and a polyA signal sequence element.

In a specific embodiment of the invention, the Cas vector has a nucleotide sequence set forth in SEQ ID NO:1 or 2.

In one embodiment of the invention, the construction method comprises co-transfecting the safe harbor site vector, the sgRNA vector, and the Cas vector into porcine cells.

In order to increase the gene editing capability of the Cas9 vector, the invention is modified on the basis of a vector purchased from addgene (Plasmid #42230,from Zhang Feng lab) pX330-U6-Chimeric _BB-CBh-hSpCas (PX 330 for short) to obtain pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO (particle pKG-GE3 for short). The map of PX330 is shown in fig. 1, modified as follows:

1) Removing redundant invalid sequences in the gRNA skeleton of the original vector;

2) Modifying a promoter: the original promoter (chicken beta-actin promoter) is modified into EF1a promoter with higher expression activity, so that the protein expression capacity of the Cas9 gene is increased;

3) Increasing the nuclear localization signal: adding a nuclear localization signal coding sequence (NLS) at the N end and the C end of the Cas9, and increasing the nuclear localization capability of the Cas 9;

4) Adding double screening markers: the original vector does not have any screening mark, is not beneficial to screening and enrichment of positive transformed cells, and P2A-EGFP-T2A-PURO is inserted into the C end of Cas9, so that the fluorescence and resistance screening capability of the vector are endowed;

5) Inserting WPRE, 3' LTR and other sequences for regulating gene expression: the WPRE, 3' LTR and other sequences are inserted into the gene frame at last, so that the protein translation capacity of the Cas9 gene can be enhanced.

The modified vector pU6gRNA-eEF1 a-mNLS-hSpCas-EGFP-PURO (called pKG-GE3 for short) has the modification site shown in figure 2, and the plasmid has the complete sequence shown in SEQ ID NO:2 is shown in the figure; the main elements of pKG-GE3 are:

1) gRNA expression element: u6 gRNA scaffold;

2) Promoter: EF1a promoter and CMV enhancer;

3) Cas9 gene comprising multiple NLSs: cas9 gene containing N-terminal and C-terminal polynuclear localization signals (NLS);

4) Screening marker genes: fluorescent and resistant double selectable marker element P2A-EGFP-T2A-PURO;

5) Element for enhancing translation: WPRE and 3' LTR enhance the translation efficiency of Cas9 and selectable marker genes;

6) Transcription termination signal: bGHpolyA signal;

7) A carrier skeleton: including Amp resistance elements and ori replicons, and the like.

The plasmid pKG-GE3 has a specific fusion gene; the specific fusion gene codes for a specific fusion protein;

The specific fusion protein sequentially comprises the following elements from the N end to the C end: two Nuclear Localization Signals (NLS), cas9 protein, two nuclear localization signals, self-cleaving polypeptide P2A, fluorescent reporter protein, self-cleaving polypeptide T2A, resistance selection marker protein;

In the plasmid pKG-GE3, the EF1a promoter is used for promoting the expression of the specific fusion gene;

in plasmid pKG-GE3, the specific fusion gene has downstream a WPRE sequence element, a 3' LTR sequence element and a bGH poly (A) signal sequence element.

The plasmid pKG-GE3 has the following elements in this order: CMV enhancer, EF1a promoter, the specific fusion gene, WPRE sequence element, 3' LTR sequence element, bGH poly (A) signal sequence element.

In the specific fusion protein, two nuclear localization signals at the upstream of the Cas9 protein are SV40 nuclear localization signals, and two nuclear localization signals at the downstream of the Cas9 protein are nucleoplasmin nuclear localization signals.

In the specific fusion protein, the fluorescent reporter protein can be EGFP protein.

In the specific fusion protein, the resistance selection marker protein may specifically be Puromycin resistance protein.

The amino acid sequence of the self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP" (the cleavage site where self-cleavage occurs is between the first amino acid residue and the second amino acid residue from the C-terminus).

The amino acid sequence of the self-cleaving polypeptide T2A is "EGRGSLLTCGDVEENPGP" (the cleavage site where self-cleavage occurs is between the first amino acid residue and the second amino acid residue from the C-terminus).

Specific fusion genes are specifically shown as SEQ ID NO:2 from nucleotide numbers 911-6706.

CMV enhancer as set forth in SEQ ID NO:2 from nucleotide 395 to 680.

The EF1a promoter is shown in SEQ ID NO:2 from nucleotide 682 to nucleotide 890.

WPRE sequence element is shown as SEQ ID NO:2 from nucleotide 6722 to nucleotide 7310.

The 3' LTR sequence element is shown in SEQ ID NO:2 from nucleotide 7382 to nucleotide 7615.

The bGH poly (A) signal sequence element is shown as SEQ ID NO:2 from nucleotide 7647 to nucleotide 7871.

Preferably, the safe harbor site vector, the sgRNA vector or the Cas vector are all circular plasmids.

In a third aspect of the invention there is provided a tissue or organ comprising a pig cell as described above.

Preferably, the tissue is breast tissue, more preferably breast epithelial tissue. Preferably, the organ is a breast.

In a fourth aspect of the present invention, there is provided a method for constructing a model pig expressing PyMT, inserting a nucleotide sequence encoding PyMT into a safe harbor site of the pig to obtain a nucleotide sequence expressing SEQ ID NO:14, a model pig of PyMT. Preferably, the swine safe harbor site is selected from swine ROSA26, AAVS1, H11 or COL1A1 safe harbor site. Further preferably, the optimal safe harbor site of the pig is a COL1A1 site.

Preferably, the construction method further comprises the step of preparing the pig cells.

Preferably, the construction method comprises transferring the pig cells into enucleated pig oocytes to obtain model pigs. In one embodiment of the invention, the engraftment site is the peri-oval space of an enucleated porcine oocyte.

In one embodiment of the present invention, the construction method comprises providing the above-mentioned pig cells or obtaining pig cells by the above-mentioned pig cell construction method, and then cloning the pig cells by somatic cell nuclear transfer animal to obtain a model pig expressing the PyMT protein.

In a fifth aspect of the present invention, a method for constructing a model pig for breast cancer is provided, wherein a nucleotide sequence encoding PyMT is inserted into a safe harbor site of the pig to obtain a nucleotide sequence expressing SEQ ID NO:14, a model pig of PyMT. Preferably, the swine safe harbor site is selected from swine ROSA26, AAVS1, H11 or COL1A1 safe harbor site. Further preferably, the optimal safe harbor site of the pig is a COL1A1 site.

In one embodiment of the present invention, the construction method comprises providing the above pig cells or obtaining pig cells by the above pig cell construction method, and then cloning the pig cells by somatic cell nuclear transfer animal to obtain a model pig of the breast cancer by homozygously or heterozygously knocking-in the pyrmt gene.

In a sixth aspect of the present invention, there is provided a safe harbor site vector comprising a nucleotide sequence encoding a PyMT and a safe harbor site vector backbone comprising a 5 'homology arm and a 3' homology arm of a safe harbor insertion site, wherein the nucleotide sequence encoding a PyMT is located between the 5 'homology arm and the 3' homology arm, and wherein the safe harbor site vector backbone is selected from any one of the following:

In a seventh aspect, the invention provides an application of the safe harbor site vector, the sgRNA vector or the sgRNA in preparation of pig cells, a model pig expressing a PyMT protein or a model pig of breast cancer.

In an eighth aspect, the present invention provides an application of the pig cell, the pig cell obtained by the construction method, and the model pig obtained by the construction method for expressing PyMT in preparing an animal model of breast cancer, or an application in screening a drug for treating breast cancer and evaluating drug efficacy, or an application in gene and cell therapy, or an application in researching pathogenesis of breast cancer.

In a ninth aspect, the present invention provides an application of the above tissue or organ or the model pig obtained by the above construction method in screening medicines for treating breast cancer and evaluating the efficacy of the medicines, or in gene and cell therapy, or in researching pathogenesis of breast cancer.

The term "vector" is a polynucleotide capable of replication under the control of itself in a cell, or a genetic element such as a plasmid, chromosome, virus, transposon, that replicates and/or is expressed by insertion into the chromosome of a host cell. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages and cosmids.

The "gRNA", also called guide RNA, described herein is an RNA that is transcribed from a sgRNA vector in a cell, is specific for a target sequence in the cell, and can form a complex with a Cas protein.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The subject (pig) of the invention has better applicability than other animals (rats, mice, primates).

Rodents such as rats and mice have great differences from humans in terms of body type, organ size, physiology, pathology and the like, and cannot truly simulate normal physiological and pathological states of humans. Studies have shown that more than 95% of drugs that are validated in mice are ineffective in human clinical trials. In the case of large animals, primates are animals with the closest relationship to humans, but are small in size, late in sexual maturity (mating begins at 6-7 years old), and single animals, the population expansion rate is extremely slow, and the raising cost is high. In addition, primate cloning is inefficient, difficult and costly.

The pig is an animal which has the closest relationship with human except primate, and has the similar body shape, weight, organ size and the like as human, and has the similar anatomical, physiological, immunological, nutritional metabolism, disease pathogenesis and the like as human. Meanwhile, the pigs are early in sexual maturity (4-6 months), have high fertility and have more piglets, and can form a larger group within 2-3 years. In addition, the cloning technology of pigs is very mature, and the cloning and feeding costs are much lower than those of primates. Pigs are thus very suitable animals as models of human diseases.

(2) Compared with the pX330 vector before transformation, the pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO (called pKG-GE3 for short) vector subjected to experimental verification in the invention replaces a stronger promoter and adds elements for enhancing protein translation, improves the expression of Cas9, increases the number of nuclear localization signals, improves the nuclear localization capability of Cas9 protein, and has higher gene editing efficiency. The invention also adds fluorescent mark and resistance mark into the carrier, which makes it more convenient to apply to the screening and enrichment of the positive transformed cells of the carrier. The efficient expression vector of Cas9 modified by the invention is adopted for gene editing, and the editing efficiency is improved by more than 100% compared with the original vector.

(3) The invention aims at the fumbling of 4 safe harbor site gene knockin expression conditions of pig genome, and selects the optimal pig genome safe harbor site for inserting exogenous genes, thereby effectively improving the expression conditions of target genes after gene knockin.

(4) The invention adopts a mammary gland specific promoter, namely a mouse mammary gland tumor virus long terminal repeated promoter (MMTV-LTR) to drive the specific expression of the exogenous oncogene in mammary gland tissues, so that the exogenous oncogene can specifically play a role in the mammary gland tissues, and meanwhile, the influence of high-level over-expression of the exogenous oncogene on organisms is avoided.

(5) The single cell clone strain homozygous and knocked in by the MMTV-PyMT expression frame can be used for carrying out somatic cell nuclear transfer animal cloning to directly obtain a cloned pig homozygous and knocked in by the MMTV-PyMT expression frame, and the homozygous inserted gene can be inherited stably. Furthermore, the method can be used in the biomedical fields such as drug screening, drug effect evaluation, gene and cell therapy, study of pathogenesis of breast cancer and the like in the next step.

In the mouse model production, fertilized eggs are generally adopted to microinjection gene editing materials and then embryo transplantation is carried out, so that the probability of directly obtaining the offspring of gene knock-in is very low (less than 1%), and meanwhile, the offspring need to be subjected to hybridization breeding to screen homozygous knock-in individuals, which is not suitable for large animal (such as pigs) model production with longer gestation period. Therefore, the method for editing and screening the positive editing single cell clone in vitro by the primary cells with high technical difficulty and high challenge is adopted, and then the corresponding model pig is directly obtained by a somatic cell nuclear transfer animal cloning technology, so that the manufacturing period of the model pig can be greatly shortened, and the manpower, material resources and financial resources are saved.

The invention obtains the PyMT model pig which is highly similar to the development process of human breast cancer through gene editing and somatic cell cloning technology, is helpful for researching and revealing the pathogenesis of breast cancer, can be used for researching drug screening, drug effect detection, gene and cell therapy and the like, can provide effective experimental data for further clinical application, and further provides a powerful experimental means for preventing and treating human breast cancer. The invention has great application value for research of pathogenesis of human breast cancer, research and development of therapeutic drugs and preclinical experiments.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a schematic diagram of the structure of plasmid pX 330.

FIG. 2 is a schematic diagram of the structure of plasmid pKG-GE 3.

FIG. 3 is a schematic diagram showing the structure of pU6gRNA vector.

FIG. 4 is a schematic representation of the insertion of a DNA molecule of about 20bp (used for transcription to form gRNA capable of binding to the target sequence) into the plasmid pKG-U6 gRNA.

FIG. 5 is a schematic representation of the structure of a fluorescent donor plasmid containing an insertion site for ROSA 26.

FIG. 6 is a schematic representation of the structure of a fluorescent donor plasmid containing an AAVS1 insertion site.

FIG. 7 is a schematic representation of the structure of a fluorescent donor plasmid containing an H11 insertion site.

FIG. 8 is a schematic structural diagram of a fluorescent donor plasmid containing COL1A1 insertion site.

FIG. 9 is a schematic structural diagram of pKG-MMTV-PyMT Donor plasmid containing COL1A1 insertion site.

FIG. 10 shows the sequencing results of the plasmid proportioning optimization test.

FIG. 11 shows the sequencing results of the editing effect of plasmid pX330 and plasmid pKG-GE 3.

FIG. 12 shows green fluorescent expression patterns of GFP regulated at different safe harbor sites.

FIG. 13 shows the results of fluorescent quantitative PCR for regulating GFP transcription level at different safe harbor sites.

FIG. 14 shows the results of FACS detection of GFP expression at different safe harbor sites.

FIG. 15 is an electropherogram for identifying whether the recombination of the MMTV-PyMT expression cassette at the 5 'end of the safe harbor insertion site of pig COL1A1 is successful, wherein WT is a wild type control, blank is a Blank, sh4 represents the safe harbor site COL1A1, lr represents the 5' homology arm, JDF represents the identification primer F, JDR represents the identification primer R,1414 or 5965 represents the detection site information.

FIG. 16 is an electrophoretogram for identifying whether the recombination of the MMTV-PyMT expression cassette at the 3 '-end of the safety harbor insertion site of porcine COL1A1 is successful, wherein WT is a wild-type control, blank is a Blank, sh4 represents the safety harbor site COL1A1, rr represents the 3' -homology arm, and 282 or 4723 represents the detection site information.

FIG. 17 is an electrophoretogram for identifying whether the MMTV-PyMT expression cassette is homozygous for insertion into the safe harbor site of porcine COL1A1, wherein WT is a wild type control, blank is a Blank, sh4 represents the safe harbor site COL1A1, JDF represents the identification primer F, JDR represents the identification primer R,1085 or 1560 represents the detection site information.

FIG. 18 shows the results of fluorescence quantitative PCR for controlling the transcript level of PyMT at the safe harbor site of the pig COL1A 1.

FIG. 19 shows the results of FACS detection of pig COL1A1 safe harbor site-regulated PyMT protein expression, wherein WT represents unmodified cloned pig mammary epithelial cells and PyMT represents mammary epithelial cells of a pig model for PyMT-expressing breast cancer prepared by nuclear transfer technology.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The recombinant plasmids constructed in the examples were all subjected to sequencing verification. Complete culture solution (% by volume): 15% fetal bovine serum (Gibco) +83% DMEM medium (Gibco) +1% Penicillin-Streptomycin (Gibco) +1% HEPES (Solarbio). Cell culture conditions: constant temperature incubator of 37 ℃,5% co ₂、5％O₂.

A method of preparing porcine primary fibroblasts: ① Taking 0.5g of pig ear tissue, removing hair and bone tissue, soaking the pig ear tissue in 75% alcohol for 30-40s, washing the pig ear tissue with PBS buffer solution containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) for 5 times, and washing the pig ear tissue with the PBS buffer solution for one time; ② Shearing the tissue with scissors, digesting with 5mL of 0.1% collagenase solution (Sigma) at 37 ℃ for 1h, centrifuging 500g for 5min, and discarding the supernatant; ③ The pellet was resuspended in 1mL of complete medium, then plated into 10-diameter cell culture dishes containing 10mL of complete medium and capped with 0.2% gelatin (VWR) and cultured to about 60% of the cell growth bottom; ④ After completion of step ③, the cells were digested with trypsin and collected, and then resuspended in complete medium for subsequent electrotransformation experiments.

Example 1 construction of vector

1. Construction of Cas9 efficient expression vector (pKG-GE 3 for short)

The commercial plasmids were: pX330-U6-Chimeric _BB-CBh-hSpCas, abbreviated as plasmid pX330, SEQ ID NO: 1.

Based on the pX330 plasmid, a plasmid pU6gRNAeEF a-mNLS-hSpCas9-EGFP-PURO, called plasmid pKG-GE3 for short, is constructed, and SEQ ID NO: 2.

Plasmid pX330 and plasmid pKG-GE3 are both circular plasmids.

The schematic structure of plasmid pX330 is shown in fig. 1.SEQ ID NO:1, nucleotides 440-725 constitute the CMV enhancer, nucleotides 727-1208 constitute the chicken β -actin promoter, nucleotides 1304-1324 encode the SV40 Nuclear Localization Signal (NLS), nucleotides 1325-5449 encode the Cas9 protein, and nucleotides 5450-5497 encode the nucleoplasmin Nuclear Localization Signal (NLS).

The schematic structure of plasmid pKG-GE3 is shown in FIG. 2.SEQ ID NO:2, nucleotides 395-680 constitute the CMV enhancer, nucleotides 682-890 constitute the EF1a promoter, nucleotides 986-1006 encode the Nuclear Localization Signal (NLS), nucleotides 1016-1036 encode the Nuclear Localization Signal (NLS), nucleotides 1037-5161 encode the Cas9 protein, nucleotides 5162-5209 encode the Nuclear Localization Signal (NLS), nucleotides 5219-5266 encode the Nuclear Localization Signal (NLS), nucleotides 5276-5332 encode the cleavage polypeptide P2A (the amino acid sequence of the cleavage polypeptide P2A is "ATNFSLLKQAGDVEENPGP", the cleavage site from the C-terminus is between the first amino acid residue and the second amino acid residue), nucleotides 5333-6046 encode the EGFP protein, nucleotides 6056-6109 encode the cleavage polypeptide T2A (the amino acid sequence of the cleavage polypeptide T2A is "EGRGSLLTCGDVEENPGP", the cleavage site from the cleavage site is between the first amino acid residue and the second amino acid residue from the C-terminal is "3782", the cleavage site from the nucleotide sequence of the cleavage polypeptide T2A is between the first amino acid residue and the second amino acid residue of the cleavage site is "^R", the cleavage site is between the nucleotide sequence of the cleavage site is No. 3 b 3, the nucleotide sequence of the cleavage site is No. 3, and the nucleotide is expressed as a position of the amino acid sequence of the cleavage element of the amino acid sequence of the first amino acid sequence is No. 3 b 3-3 b 3, and No. 3 b 3 is expressed. SEQ ID NO:2, 911-6706 form a fusion gene, expressing a fusion protein. Due to the presence of self-cleaving polypeptides P2A and T2A, the fusion protein spontaneously cleaves into three separate proteins, cas9 protein, EGFP protein and Puro resistant protein.

Compared with the plasmid pX330, the constructed plasmid pKG-GE3 is mainly modified as follows: ① Removing residual gRNA backbone sequences (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTT) to reduce interference; ② The original chicken beta-actin promoter is modified into an EF1a promoter with higher expression activity, so that the protein expression capacity of the Cas9 gene is increased; ③ Adding nuclear localization signal coding genes (NLS) at the upstream and downstream of the Cas9 gene, and increasing the nuclear localization capability of the Cas9 protein; ④ The original plasmid has no eukaryotic cell screening mark, is not beneficial to screening and enriching positive transformed cells, and sequentially inserts P2A-EGFP-T2A-PURO coding genes at the downstream of Cas9 genes, so that eukaryotic cell fluorescence and puromycin resistance double screening marks are endowed; ⑤ The insertion of the WPRE element and the 3' ltr sequence element enhances the protein translation capacity of the Cas9 gene.

2. Construction of pKG-U6gRNA expression vector

Constructing a pKG-U6gRNA vector by taking pUC57 as a starting plasmid, wherein the structure schematic diagram is shown in FIG. 3, and the sequence is shown in SEQ ID NO: 3. SEQ ID NO:3, nucleotides 2280 to 2539 constitute the hU6 promoter and nucleotides 2558 to 2637 are used for transcription to form the gRNA backbone. When in use, a DNA molecule (target sequence binding region for transcription to form gRNA) of about 20bp is inserted into plasmid pKG-U6gRNA to form a recombinant plasmid, the schematic diagram is shown in FIG. 4, and the recombinant plasmid is transcribed in cells to obtain gRNA.

3. Construction of different safe harbor site Donor vectors containing GFP Gene

Plasmids PB-1G 2R 3-puro-ROSA26, PB-1G 2R 3-puro-AAVS1, PB-1G 2R 3-puro-H11 and PB-1G 2R3-puro-COL1A1 were constructed.

The structural schematic diagram of the plasmid PB-1G 2R 3-puro-ROSA26 is shown in FIG. 5.SEQ ID NO: in 4, nucleotides 1 to 345 constitute the swine genome region 5 'of the ROSA26 safety harbor insertion site (SH 1 left arm is shown as SEQ ID NO: 5), nucleotides 9184 to 10195 constitute the swine genome region 3' of the ROSA26 safety harbor insertion site (SH 1 right arm is shown as SEQ ID NO: 6), nucleotides 346 to 546, 3132 to 3531, 6506 to 6706, 8975 to 9175 constitute 4 different insulator regions, nucleotides 1954 to 3131 constitute the EF-1 alpha promoter, nucleotides 1216 to 1935 encode the EGFP protein, nucleotides 637 to 1209 constitute the EF-1 alpha poly (A) signal, nucleotides 3543 to 4042 constitute the PGK promoter, nucleotides 4059 to 4769 encode the mCherry protein, nucleotides 4791 to 5015 constitute the bGH (A) signal, nucleotides 5054 to 6504 constitute the loxP-ro-loxP-region, and nucleotides 7259 to 7269 constitute the poly (pC) signal.

The structural schematic diagram of the plasmid PB-1G 2R 3-puro-AAVS1 is shown in FIG. 6. Only SEQ ID NO:4 with the 5' end porcine genomic region (SH 2 left arm) of AAVS1 safe harbor insertion site, see SEQ ID NO:7, preparing a base material; setting SEQ ID NO:4 by replacing nucleotides 9184-10195 in AAVS1 safe harbor insertion site 3' end porcine genomic region (SH 2 right arm), see SEQ ID NO:8. other sequences and SEQ ID NO:4 are consistent.

The structural schematic diagram of the plasmid PB-1G 2R 3-puro-H11 is shown in FIG. 7. Only SEQ ID NO:4 by replacing nucleotide 1-345 in the genome region of the pig 5' to the H11 safe harbor insertion site (SH 3 left arm), see SEQ ID NO:9, a step of performing the process; setting SEQ ID NO:4 by replacing nucleotide 9184-10195 in the sequence of the 3' -end pig genome region (SH 3 right arm) of the H11 safe harbor insertion site, see SEQ ID NO:10. other sequences and SEQ ID NO:4 are consistent.

The structural schematic diagram of the plasmid PB-1G 2R 3-puro-COL1A1 is shown in FIG. 8. Only SEQ ID NO:4 by substituting nucleotide 1-345 in COL1A1 safe harbor insertion site 5' end porcine genomic region (SH 4 left arm), see SEQ ID NO:11; setting SEQ ID NO:4 by replacing nucleotides 9184-10195 in the sequence of SEQ ID NO:12. other sequences and SEQ ID NO:4 are consistent.

4. Construction of pKG-MMTV-PyMT Donor vector

The construction of plasmid pKG-MMTV-PyMT is shown in FIG. 9.SEQ ID NO:13, nucleotide numbers 1-852 are homologous sequences at the 5' end of a safety harbor insertion site of a pig genome COL1A1, nucleotide numbers 879-1079 are sequences of an Insulator1 (Insulator 1), nucleotide numbers 1080-2394 are sequences of an MMTV-LTR promoter (from pGL4.36[ luc2P MMTV Hygro ] plasmid, the sequence is shown as SEQ ID NO:15, purchased from Shanghai N.Y. Biotechnology Co., ltd.), nucleotide numbers 2407-3672 are sequences of coding PyMT (total gene synthesis is carried out in a biological organism, the coded amino acid sequence is shown as SEQ ID NO: 14), nucleotide numbers 3721-3945 are sequences of bGHO (A), nucleotide numbers 4107-4436 are sequences of an SV40 promoter, nucleotide numbers 4485-5081 are sequences of coding sequences of a resistance protein (abbreviated as Puro ^R protein), nucleotide numbers 5261-5382 are sequences of an SV40 Poly (A) and nucleotide numbers 5431-5460 are sequences of a 5669-563, respectively, and the nucleotide numbers 5469 are sequences of the safety harbor 1-561.

Example 2 comparison of the effects of plasmid pX330 and plasmid pKG-GE3

Selecting a high-efficiency gRNA target located in the RAG1 gene:

target for RAG1-gRNA 4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO: 16).

Primers used to amplify the fragments containing the target were as follows:

RAG1-nF126：5’-CCCCATCCAAAGTTTTTAAAGGA-3’(SEQ ID NO：17)；

RAG1-nR525：5’-TGTGGCAGATGTCACAGTTTAGG-3’(SEQ ID NO：18)。

Porcine primary fibroblasts were prepared from ear tissue of a junior river-flavored pig (female, blood group AO).

1. Construction of RAG1 Gene gRNA recombinant plasmid

Plasmid pKG-U6gRNA was digested with restriction enzyme BbsI, and the vector backbone (about 3kb linear fragment) was recovered. RAG1-4S and RAG1-4A were synthesized separately, and then mixed and annealed to give a double-stranded DNA molecule having cohesive ends. The double-stranded DNA molecule having a cohesive end and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (RAG 1-gRNA 4).

RAG1-4S：5’-caccgAGTTATGGCAGAACTCAGTG-3’(SEQ ID NO：19)；

RAG1-4A：5’-aaacCACTGAGTTCTGCCATAACTc-3’(SEQ ID NO：20)。

RAG1-4S and RAG1-4A are single stranded DNA molecules.

2. Plasmid proportioning optimization

1. Plasmid cotransfection of porcine primary fibroblasts

A first group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.44. Mu.g plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.56. Mu.g of plasmid pKG-GE3. Namely, the molar ratio of the plasmid pKG-U6gRNA (RAG 1-gRNA 4) to the plasmid pKG-GE3 is as follows: 1:1.

Second group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.72. Mu.g plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.28. Mu.g of plasmid pKG-GE3. Namely, the molar ratio of the plasmid pKG-U6gRNA (RAG 1-gRNA 4) to the plasmid pKG-GE3 is as follows: 2:1.

Third group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.08 μg of plasmid pKG-GE3. Namely, the molar ratio of the plasmid pKG-U6gRNA (RAG 1-gRNA 4) to the plasmid pKG-GE3 is as follows: 3:1.

Fourth group: plasmid pKG-U6gRNA (RAG 1-gRNA 4) was transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4).

Co-transfection was performed by electric shock transfection using a mammalian nuclear transfection kit (Neon kit, thermofisher) and a Neon TM transfection system electrotransfection apparatus (parameters set to 1450V, 10ms, 3 pulses).

2. After the step 1 is completed, the culture is carried out for 16 to 18 hours by adopting the complete culture solution, and then the culture is carried out by replacing the new complete culture solution. The total incubation time was 48 hours.

3. After step2 is completed, cells are digested and collected by trypsin, genomic DNA is extracted, PCR amplification is performed by using a primer pair consisting of RAG1-nF126 and RAG1-nR525, and then electrophoresis is performed.

The band of interest was recovered after electrophoresis and sequenced, and the sequencing results are shown in FIG. 10.

The editing efficiency of different targets is obtained by analyzing the sequencing peak diagram by using Synthego ICE tools. The gene editing efficiency of the first group to the third group was 9%, 53%, 66% in this order. The fourth group did not undergo gene editing. The results show that the third group has the highest editing efficiency, and the optimal ratio of the single gRNA plasmid to the Cas9 plasmid is determined to be 3:1, and the actual use amount of the plasmid is 0.92 mug to 1.08 mug.

3. Comparison of the effects of plasmid pX330 and plasmid pKG-GE3

1. Co-transfection

RAG1-B group: plasmid pKG-U6gRNA (RAG 1-gRNA 4) was transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4).

RAG1-330 group: plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pX330 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.08. Mu.g of plasmid pX330, i.e.the molar ratio of the two DNA is 3:1.

RAG1-KG group: the plasmid pKG-U6gRNA (RAG 1-gRNA 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (RAG 1-gRNA 4): 1.08. Mu.g of plasmid pKG-GE3, i.e.the molar ratio of the two DNA was 3:1.

3. After step 2 is completed, cells are digested and collected by trypsin, genomic DNA is extracted, PCR amplification is carried out by using a primer pair consisting of RAG1-nF126 and RAG1-nR525, and the products are sequenced.

The editing efficiency of different targets is obtained by analyzing the sequencing peak diagram by using Synthego ICE tools. No gene editing occurred in RAG1-B groups. The editing efficiency of RAG1-330 groups and RAG1-KG groups is 28% and 68% in sequence. Exemplary peak diagrams of sequencing results are shown in FIG. 11. The results show that the use of plasmid pKG-GE3 results in a significant increase in gene editing efficiency compared to the use of plasmid pX 330.

Example 3 screening of pig genome optimal safe harbor site for site-directed insertion of exogenous Gene

1. Construction of pig genome ROSA26, AAVS1, H11 and COL1A1 safe harbor site gRNA recombinant vector and efficient cutting target spot screening

Through the early screening, the efficient cleavage targets of the ROSA26, H11, AAVS1 and COL1A1 safe harbor sites are respectively the sgRNA _ROSA26-g3 (cleavage efficiency 38%), the sgRNA _AAVS1-g4 (cleavage efficiency 30%), the sgRNA _H11-g1 (cleavage efficiency 60%), the sgRNA _COL1A1-g3 (cleavage efficiency 56%), and the target sequences are as follows:

sgRNA _ROSA26-g3 target: 5'-GAAGGAGCAAACTGACATGG-3' (SEQ ID NO: 21);

sgRNA _AAVS1-g4 target: 5'-TGCAGTGGGTCTTTGGGGAC-3' (SEQ ID NO: 22);

sgRNA _H11-g1 target: 5'-TTCCAGGAACATAAGAAAGT-3' (SEQ ID NO: 23);

sgRNA _COL1A1-g3 target: 5'-GCAGTCTCAGCAACCACTGA-3' (SEQ ID NO: 24).

The gRNA plasmids corresponding to the 4 gRNA targets are pKG-U6gRNA (ROSA 26-g 3), pKG-U6gRNA (AAVS 1-g 4), pKG-U6gRNA (H11-g 1) and pKG-U6gRNA (COL 1A1-g 3), wherein the backbone vectors are pKG-U6gRNA (SEQ ID NO: 3), and the plasmid construction method is the same as in example 2.

2. Fluorescent Donor vector containing homology arms on both sides of insertion site of different safety harbors (i.e., vector of different safety harbors containing foreign gene GFP), sgRNA vector and Cas9 vector (pKG-GE 3 prepared in example 1) were mixed with electric pig primary fibroblast

And respectively co-transfecting the PB-1G 2R 3-puro-different safe harbor insertion site fluorescent vectors with the corresponding high-efficiency sgRNA vectors and the high-efficiency Cas9 vectors into porcine primary fibroblasts. Electrotransfection experiments (parameters set to 1450V, 10ms, 3 pulses) were performed using a mammalian nuclear transfection kit (Neon kit, thermofisher) with a Neon TM transfection system electrometer.

Co-transfection plasmid combination and ratio:

A first group: the plasmid PB-1G 2R 3-puro-ROSA26, plasmid pKG-U6gRNA (ROSA 26-g 3) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 1.26. Mu.g of plasmid PB-1G 2R 3-puro-ROSA26: 0.82. Mu.g plasmid pKG-U6gRNA (ROSA 26-g 3): 0.92. Mu.g of plasmid pKG-GE3, i.e.3 DNA molar ratios: 1:3:1.

Second group: the plasmid PB-1G 2R 3-puro-AAVS1, plasmid pKG-U6gRNA (AAVS 1-g 4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 1.26. Mu.g of plasmid PB-1G 2R 3-puro-AAVS1: 0.82. Mu.g plasmid pKG-U6gRNA (AAVS 1-g 4): 0.92. Mu.g of plasmid pKG-GE3, i.e.3 DNA molar ratios: 1:3:1.

Third group: the plasmid PB-1G 2R 3-puro-H11, plasmid pKG-U6gRNA (H11-g 1) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 1.26. Mu.g of plasmid PB-1G 2R 3-puro-H11: 0.82. Mu.g plasmid pKG-U6gRNA (H11-g 1): 0.92. Mu.g of plasmid pKG-GE3, i.e.3 DNA molar ratios: 1:3:1.

Fourth group: the plasmid PB-1G 2R 3-puro-COL1A1, plasmid pKG-U6gRNA (COL 1A1-g 3) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 1.26. Mu.g of plasmid PB-1G 2R 3-puro-COL1A1: 0.82. Mu.g of plasmid pKG-U6gRNA (COL 1A1-g 3): 0.92. Mu.g of plasmid pKG-GE3, i.e.3 DNA molar ratios: 1:3:1.

Fifth group: pig primary fibroblast, the same electrotransformation parameters do not add any plasmid for electrotransformation operation.

The specific implementation method comprises the following steps:

And (3) cells: the fusion degree of primary fibroblasts of pigs before electrotransformation reaches 60%, trypsin digestion is performed at 0.25%, trypan blue staining is counted, and five groups of electrotransformation are performed on equal cells.

Pig primary cell electrotransformation:

(1) Cells were digested with pancreatin, the resulting cell suspension was washed once with PBS phosphate buffer (Solarbio), centrifuged for 6min at 600g, the supernatant was discarded, and cells (11 μl/min) were resuspended using 58 μl of electrotransfer base solution rbuffer, while avoiding air bubbles during resuspension;

(2) Sucking 10 mu L of cell suspension and plasmid electrotransformation reaction liquid, and uniformly mixing, wherein no bubbles are generated in the mixing process;

(3) Placing an electric rotating cup with a reagent cassette in a Neon TM transfection system electric rotating instrument cup groove, and adding 3mL Buffer E;

(4) Sucking 10 mu L of the mixed solution obtained in the step 2) by using an electrotransfer gun, inserting the mixed solution into a electric shock cup, selecting an electrotransfer program (1450V 10ms 3pulse), transferring the mixed solution in the electrotransfer gun into a 6-well plate immediately after electric shock transfection, wherein each well contains 3mL of complete culture solution (15% fetal bovine serum (Gibco) +83% DMEM medium (Gibco) +1% P/S (Gibco Penicillin-Streptomycin) +1% HEPES (Solarbio));

(5) Mixing, and culturing in a constant temperature incubator at 37deg.C and 5% CO ₂、5％O₂;

(6) And (3) transferring the liquid for 12-24 hours, pressurizing the liquid for 48 hours by using puromycin, and screening positive cells.

3. Puromycin pressurized screening and cell GFP fluorescence intensity detection

Cells were electrotransformed with plasmid for 48h, 1.5 μg/mL puromycin was added for selection, medium containing puromycin at the same concentration was changed every two days while GFP green fluorescence photographing was performed, selection was continued for two weeks, and pressure selection was continued for one week after intracellular plasmid was completely degraded. Judging the efficiency of expressing exogenous genes at the safe harbor site through the fluorescence expression intensity of GFP.

After puromycin is screened for one week, the fluorescent intensity of the ROSA26 and COL1A1 safe harbor site experimental group is obviously stronger than that of an AAVS1 and H11 experimental group; after two weeks of puromycin screening, the fluorescence intensities are sequentially from strong to weak: COL1A1> ROSA26> H11> AAVS1, wherein the fluorescence intensity of the H11 group is not uniform, the overall fluorescence intensity of the ROSA26 group is uniform, the fluorescence intensity is high, the fluorescence expression of the AAVS1 group cells is weakest, the number of the COL1A1 group fluorescent cells is the largest, and the fluorescence is the strongest; after puromycin is continuously screened for three weeks, the fluorescence intensity is sequentially from strong to weak:

COL1A1> ROSA26> H11> AAVS1, the results are shown in FIG. 12.

4. GFP gene transcription level assay

To compare the differences in mRNA transcription levels after GFP gene integration into four different safe harbor sites, it was possible to participate in the regulation of GFP expression and the effect on expression levels. Designing a pair of primers at the exon of GFP gene, screening cells three weeks later by puromycin, extracting total RNA, reversely transcribing into cDNA, and detecting the transcription level of the primary cells after the GFP gene is integrated at four different safe harbor sites, and simultaneously using the wild primary cells as a control. GAPDH was used as a reference gene and calculated according to the method of 2 ^-ΔCt.

(1) Primer information (Table 1)

Table 1: fluorescent quantitative PCR primer information

(2) Total RNA extraction from cells

Total cellular RNA extraction according to Simply P Total RNA extraction kit of Bio Flux

(3) First strand cDNA acquisition

Reverse transcription kit according to VazymeII 1st Strand cDNASynthesis Kit (R211-01/02) Synthesis of cDNA according to the instruction

The first chain, specific steps and procedure are as follows:

1) Preparing first strand cDNA synthesis reaction liquid

The following mixture in Table 2 was prepared in an RNase-free centrifuge tube

TABLE 2

Gently beating and mixing by a pipetting gun.

2) The first strand cDNA synthesis reaction was performed under the following conditions, and the reaction conditions are shown in Table 3.

TABLE 3 Table 3

The product is immediately used for qPCR reaction or stored at-80 ℃ to avoid repeated freezing and thawing.

(4) Fluorescent quantitative PCR

Detection of insertion of four different sets of safe harbor sites (ROSA 26, AAVS1, H11, COL1A 1) by real-time fluorescent quantitative PCR

GFP expression level in porcine primary fibroblasts, GAPDH was used as an internal reference gene. The operation steps and the program are as follows:

1) The preparation of the reaction system is shown in Table 4

TABLE 4 Table 4

2) QPCR reaction procedure is shown in Table 5 below

TABLE 5

3) Statistics and analysis

Data analysis was performed using SPSS statistical software, expressed as (mean ± standard deviation), and statistical analysis was performed using a two-factor analysis of variance. The results of 2 ^-ΔCt values show that the GFP expression level of the AAVS1 and H11 groups is lower after three weeks of puromycin screening, the GFP expression level of the ROSA26 and COL1A1 groups is higher, the difference of the GFP transcription levels of the COL1A1 group and the ROSA26 group relative to the AAVS1 and H11 groups is extremely obvious (P < 0.01), the values of 2 ^-ΔCt are shown in table 6, and the analysis result of the difference significance is shown in figure 13.

Table 6:2 ^-ΔCt value information

In summary, from the results of fluorescent signal intensity and GFP gene real-time fluorescent quantitative PCR after culturing cells for three weeks, it can be concluded that among four genomic safe harbor sites of ROSA26, AAVS1, H11, and COL1A1, the COL1A1 site has the best expression effect after insertion of foreign gene.

5. FACS detection of protein expression level of GFP Gene

To compare the expression of GFP after the GFP gene was integrated into four different safety harbor sites. Cells were digested with trypsin, centrifuged at 400g for 4min, and the supernatant was discarded. Cells were resuspended in 1mL of medium and the cell suspensions were transferred into flow tubes, respectively. GFP signals were detected in the FITC channel of BD FACSMelody flow cytometer and 5X 10 ⁴ cells were collected for analysis with wild type cells as negative control, as shown in FIG. 14. The results show that GFP fluorescence signal COL1A1> ROSA26> H11> AAVS1.

Thus, in summary of the above results, the COL1A1 site was the pig primary cell safe harbor site that most efficiently expressed the exogenous gene among the four safe harbor sites of ROSA26, AAVS1, H11, COL1 A1.

EXAMPLE 4 preparation of a monoclonal clone with MMTV-PyMT expression cassette site-directed insertion into the safe harbor site of porcine COL1A1

1. Co-transfection

The plasmid pKG-U6gRNA (COL 1A1-g 3), plasmid pKG-GE3 and plasmid pKG-MMTV-PyMT (as shown in SEQ ID NO: 13) were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.89. Mu.g of plasmid pKG-U6gRNA (COL 1A1-g 3): 0.99. Mu.g plasmid pKG-GE3: 1.12. Mu.g of plasmid pKG-MMTV-PyMT, 3 DNA molar ratios: 3:1:1.

2. Puromycin pressure screening

1. Puromycin screening MMTV-PyMT expression cassette positive insert cells

After cells are subjected to plasmid electrotransformation for 48 hours, 1.5 mug/mL puromycin is added for screening, the culture medium containing puromycin with the same concentration is replaced every day, and after continuous screening for one week, all wild control cells die, and a large number of cells die after one week of plasmid electrotransformation screening of pKG-MMTV-PyMT due to lower electrotransformation efficiency; the puromycin is added continuously for screening for one week, cells only die sporadically, part of positive clones start to divide and proliferate, and the number of cells is increased continuously; the pressure screening was continued for one week to completely degrade the intracellular plasmid to exclude false positive cell clones. After three weeks of pressure screening, the pressure is stopped, and after two generations of culture, the cell state is recovered, the cell is used for monoclonal sorting.

2. Monoclonal sorting and amplifying culture

(1) Screening the puromycin for three weeks, carrying out monoclonal separation, carrying out digestion by using trypsin, neutralizing by using a complete culture medium, centrifuging for 5min by 500g, removing supernatant, re-suspending sediment by using 1mL of the complete culture medium, properly diluting, picking single cells by using an oral suction tube, transferring the single cells into a 96-well plate containing 100 mu L of the complete culture medium, picking one 96-well single cell per group of cells, placing each cell into a constant temperature incubator with 37 ℃ and 5% CO ₂、5％O₂ for culture, changing the cell culture medium (containing 1.5% puromycin) every 2-3 days, observing the growth condition of each cell by using a microscope during the period, and excluding the cell-free and non-single cell clone wells;

(2) After the wells of the 96-well plate were full of cells (about 2 weeks), cells were digested with trypsin and collected, 2/3 of the cells were inoculated into 6-well plates containing complete medium, and the remaining 1/3 of the cells were collected in 1.5mL centrifuge tubes for the next genotyping;

(3) When 6-well plate cells were grown to 50% confluence, they were digested with 0.25% (Gibco) trypsin and harvested, and frozen using cell cryopreservation solution (90% complete medium+10% DMSO, volume ratio).

3. Single cell clone genome level identification of pig COL1A1 safety harbor site fixed point inserted MMTV-PyMT expression frame

To examine whether the pig COL1A1 safe harbor site was successfully site-directed inserted into the MMTV-PyMT expression cassette. Taking single cell clone after puromycin pressurized screening, extracting genome DNA, performing PCR amplification (respectively adopting a primer pair formed by sh4-Lr-JDF1414 and sh4-Lr-JDR5965, a primer pair formed by sh4-Rr-JDF282 and sh4-Rr-JDR4723, and a primer pair formed by sh4-wt-JDF1085 and sh4-wt-JDR 1560), and then performing electrophoresis. Porcine primary adipose stem cells were used as wild-type controls. The primer pair consisting of sh4-Lr-JDF1414 and sh4-Lr-JDR5965 is used for identifying whether the MMTV-PyMT expression frame at the 5' end of the porcine COL1A1 safety harbor insertion site is successfully recombined; the primer pair consisting of sh4-Rr-JDF282 and sh4-Rr-JDR4723 is used for identifying whether the MMTV-PyMT expression frame at the 3' -end of the porcine COL1A1 safety harbor insertion site is successfully recombined; the primer pair consisting of sh4-wt-JDF1085 and sh4-wt-JDR1560 is used for identifying whether the MMTV-PyMT expression cassette for site-specific insertion of the safety harbor site of the pig COL1A1 is homozygous or heterozygous.

sh4-Lr-JDF1414：CCTGCTGTAAGTGCCGTAGT(SEQ ID NO：29)

sh4-Lr-JDR5965：CTAGGGGCACAGCACGTC(SEQ ID NO：30)

sh4-Rr-JDF282：AAGTTATTAGGTCTGAAGAGGAGTTT(SEQ ID NO：31)

sh4-Rr-JDR4723:CCCATCATTCCGTCCCAGAG(SEQ ID NO：32)

sh4-wt-JDF1085:TGCTGAGTTCTGGCTTCCTG(SEQ ID NO：33)

sh4-wt-JDR1560:TCTACCAAGAGAGTGACCAGCAG(SEQ ID NO：34)

The electrophoresis patterns are shown in fig. 15, 16 and 17, respectively. From the results of electrophoresis, we preliminarily determined that single cell clones numbered 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 were clones that successfully inserted MMTV-PyMT at the safe harbor site of pig COL1A1, with single cell clones numbered 6, 10 being homozygous site-specific insertion and single cell clones numbered 1,2,3,4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 being heterozygous site-specific insertion (table 7).

TABLE 7 genotype of single cell clone of pig COL1A1 safety harbor site inserted with MMTV-PyMT expression cassette at fixed point

4. Cloning and producing breast cancer model pig by somatic cell nuclear transfer technology

1. Oocyte in vitro maturation

Pig ovaries were first taken from slaughter houses and oocyte collection and In Vitro Maturation (IVM) culture were performed. Cumulus oocyte complexes (Cumulus-oocyte complexes, COCs) were withdrawn from follicles 3 to 6mm in diameter. COCs with at least three layers of dense cumulus cells were selected and approximately 300-400 COCs were cultured in four-well plates containing IVM medium, wherein each well contained 200 μl of IVM medium for approximately 50 COCs. The COCs-containing plates were incubated at 38.5 ℃ in an incubator with 5% co ₂ and saturated humidity for 42-44 hours.

2. Somatic Cell Nuclear Transfer (SCNT) and embryo transfer

The SCNT technical scheme is as follows: the cultured COCs were treated with 0.1% (w/v) hyaluronidase to remove cumulus cells. The first polar body and adjacent cytoplasm (containing the oocyte nucleus) in the perioval gap was removed by gentle aspiration in TLH-PVA solution using a beveled glass needle. Positive fibroblasts homozygous for MMTV-PyMT were injected into the peri-oval space of the enucleated oocyte. Embryos are reconstituted in a fusion medium (0.25 MD-sorbitol, 0.05mM Mg (C ₂H₃O₂)₂, 20Mg/mLBSA and, 0.5mM HEPES [ acid-free ]) using an electrofusion apparatus (LF 201, japanese NEPA Gene Co., ltd.) with a single direct current pulse fusion of 200V/mM for 20. Mu.s, then the embryos are cultured in PZM-3 for 0.5-1h, and then the embryos are activated with a single pulse of 150V/mM in an activation medium containing 0.25 MD-sorbitol ,0.01mM Ca(C₂H₃O₂)₂,0.05mM Mg(C₂H₃O₂)₂ and 0.1Mg/mLBSA for 100ms. The embryos are placed in a PZM-3 solution containing 5. Mu.g/mL cytochalasin B, equilibrated for 2h in an incubator with a saturated humidity of 38.5℃and 5% CO ₂、5％O₂ and 90% N ₂, and then cultured in PZM-3 medium under the same culture conditions as described above until embryo transplantation.

SCNT embryos were transferred into oviducts of recipient sows. About 23 days after embryo transfer, pregnancy was confirmed using an ultrasonic scanner (HS-101V, toyota Kogyo Co., japan), and cloned piglets were born at 116-117 days. A total of 7 breast cancer model pigs were produced by 4 successfully pregnant sows.

5. Transcriptional level detection of pig PyMT gene of breast cancer model

In order to detect whether a model pig with COL1A1 safety harbor site inserted into an MMTV-PyMT expression frame can express mRNA of a PyMT gene or not, a pair of primers is designed in the MMTV-PyMT expression frame, mammary tissues of a PyMT model cloned pig and a non-modified control cloned pig (with the same cell source) are separated, total RNA is extracted, and the total RNA is reversely transcribed into cDNA for detecting the mRNA expression level of the PyMT gene of pig mammary cells, and meanwhile, the non-modified cloned pig mammary cells (called WT cells) are used as a control. The calculation was performed according to the method of 2 ^-ΔCt using beta-actin as a reference gene. For detailed procedures, reference is made to example 3 (IV, GFP gene transcription level assay).

Primer information is shown in Table 8:

TABLE 8 fluorescent quantitative PCR primer information

Data analysis was performed using SPSS statistical software, expressed as (mean ± standard deviation), and statistical analysis was performed using one-way analysis of variance. The results of the values of 2 ^-ΔCt show that the expression level of the PyMT gene of the modified pig mammary cells is significantly higher than that of the unmodified cloned pig mammary cells (figure 18).

In summary, according to the result of real-time fluorescence quantitative PCR, the PyMT gene is remarkably expressed in mammary cells of the modified breast cancer model pig.

6. FACS detection of protein expression level of pig PyMT gene in breast cancer model

To compare the expression of the PyMT gene in modified and unmodified porcine mammary cells. Mammary gland tissues of model pigs and control pigs are separated respectively, put into digestion liquid containing 250U/mL collagenase I, 150U/mL hyaluronidase, 1% penicillin and 1% streptomycin, shake digested for 1h at 37 ℃, and then the digestion liquid is neutralized by an equal volume of complete culture medium (DMEM/F12 cell culture liquid containing 10% FBS) and respectively pass through 100 mu m and 40 mu m cell sieves to obtain single mammary gland epithelial cells. Cells were washed with PBS, centrifuged, and the supernatant was discarded. 90% pre-chilled methanol at-20deg.C was added to fully re-suspend the cells, and the cells were fixed for 20min. After the fixation, centrifuging and discarding the fixation solution. Blocking was performed by adding 3% BSA for 1h. After the end of the sealing, centrifuging and discarding the sealing liquid. And the cells were resuspended in a specific dilution of the PyMT antibody (Abcam, ab 15085) after washing with complete medium (final antibody concentration 1:200 dilution) and incubated for 2h at room temperature. After the antibody incubation was completed, after washing with complete medium, goat anti-rat secondary antibody (Abcam, ab 150157) was added at a final concentration of 1: after 1000 dilution, incubation for 1h at room temperature, followed by extensive washing with complete medium, 500 μl of complete medium was added to resuspend the cells and the cell suspension was transferred into a flow tube. The PyMT antibody fluorescent signal was detected in the FITC channel of BD FACSMelody flow cytometer and 5×10 ⁴ cells were collected for analysis, the results are shown in fig. 19. The results showed that the antibody fluorescence signal of PyMT was clearly detected in mammary epithelial cells (PyMT) of pig COL1A1 safety harbor site-directed insertion MMTV-PyMT, whereas no antibody fluorescence signal of PyMT was detected in wild-type mammary epithelial cells (WT) of control pigs, indicating a higher expression of inserted PyMT in mammary epithelial cells of breast cancer model pigs, and further indicating that breast cancer model pigs were successfully constructed.

Furthermore, the breast cancer model pig prepared by the application can be used in the biomedical fields such as drug screening, drug effect evaluation, gene and cell therapy, study of pathogenesis of breast cancer and the like in the next step.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Sequence listing

<110> Nanjing Kidney Gene engineering Co., ltd

<120> Construction method and application of breast cancer model pig

<130> 1

<160> 43

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8484

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360

agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420

aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480

ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720

ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780

ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840

ggggggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 900

agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 960

aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct 1020

ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga 1080

gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta 1140

agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca 1200

ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 1260

ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa 1320

ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg 1380

caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt 1440

caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct 1500

gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata 1560

caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa 1620

ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa 1680

gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta 1740

ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg 1800

gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg 1860

cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta 1920

caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct 1980

gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga 2040

gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt 2100

caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga 2160

cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc 2220

cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat 2280

caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct 2340

gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt 2400

cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt 2460

ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa 2520

gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca 2580

ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt 2640

cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt 2700

gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac 2760

catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat 2820

cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 2880

cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 2940

gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 3000

gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 3060

cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 3120

cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 3180

cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 3240

cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 3300

gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 3360

caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 3420

cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 3480

ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 3540

cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 3600

ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 3660

gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 3720

cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 3780

cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 3840

gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 3900

cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc 3960

cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 4020

tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 4080

taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 4140

acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 4200

agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 4260

ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 4320

cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 4380

ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 4440

taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 4500

ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 4560

cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 4620

gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc 4680

caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 4740

cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 4800

caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 4860

cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 4920

ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 4980

aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata 5040

tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 5100

tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 5160

gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 5220

cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 5280

gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga 5340

ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat 5400

caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc 5460

ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga 5520

tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 5580

tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 5640

tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 5700

ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg 5760

atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 5820

gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 5880

ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 5940

atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6000

tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt 6060

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6120

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 6180

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6240

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct 6300

cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg 6360

agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 6420

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 6480

caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6540

ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6600

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6660

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6720

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6780

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6840

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6900

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6960

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 7020

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 7080

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 7140

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 7200

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 7260

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 7320

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 7380

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 7440

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 7500

ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc 7560

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7620

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7680

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7740

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7800

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7860

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7920

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7980

ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 8040

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 8100

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 8160

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 8220

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 8280

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 8340

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 8400

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 8460

tttgctggcc ttttgctcac atgt 8484

<210> 2

<211> 10476

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tagcgcgtgc 360

gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc 420

cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga 480

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 540

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 600

ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 660

tagtcatcgc tattaccatg ggggcagagc gcacatcgcc cacagtcccc gagaagttgg 720

ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa 780

gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 840

cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggttggac 900

cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta 960

caaagacgat gacgataaga tggcccccaa aaagaaacga aaggtgggtg ggtccccaaa 1020

gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg 1080

cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc 1140

cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat 1200

cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc 1260

cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa 1320

cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga 1380

agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta 1440

ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa 1500

ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt 1560

cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct 1620

ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc 1680

caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca 1740

gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct 1800

gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa 1860

ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga 1920

cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt 1980

gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca 2040

ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa 2100

agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag 2160

ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga 2220

actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg 2280

cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga 2340

tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat 2400

cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa 2460

gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc 2520

ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct 2580

gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa 2640

atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat 2700

cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta 2760

cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa 2820

cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga 2880

caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga 2940

cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat 3000

gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa 3060

cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt 3120

cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat 3180

ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc 3240

cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt 3300

gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca 3360

gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat 3420

caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa 3480

cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact 3540

ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa 3600

ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga 3660

caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa 3720

cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct 3780

gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac 3840

aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa 3900

gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa 3960

ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta 4020

cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt 4080

cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga 4140

aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac 4200

cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga 4260

aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag 4320

catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga 4380

gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc 4440

taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa 4500

agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat 4560

catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa 4620

agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa 4680

cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct 4740

gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc 4800

ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga 4860

gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga 4920

caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa 4980

tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga 5040

caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat 5100

ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga 5160

caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gcggctccaa 5220

gcggcctgcc gcgacgaaga aagcgggaca ggccaagaaa aagaaaggat ccggcgcaac 5280

aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa 5340

gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 5400

cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 5460

cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 5520

cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 5580

cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 5640

cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 5700

cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 5760

caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 5820

gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 5880

gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 5940

ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 6000

cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggct ccggcgaggg 6060

caggggaagt cttctaacat gcggggacgt ggaggaaaat cccggcccaa ccgagtacaa 6120

gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc 6180

cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg 6240

ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 6300

ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 6360

ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 6420

gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 6480

ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 6540

ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 6600

ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc 6660

cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga 6720

caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 6780

tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 6840

tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 6900

gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 6960

tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 7020

tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 7080

gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 7140

cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 7200

caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 7260

tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta 7320

agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 7380

ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 7440

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 7500

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 7560

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc 7620

cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 7680

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 7740

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 7800

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 7860

gggctctatg gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 7920

atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 7980

cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 8040

ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 8100

taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 8160

aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 8220

ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 8280

tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt 8340

ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 8400

ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 8460

cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 8520

cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 8580

caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8640

tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8700

ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8760

gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8820

cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8880

tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8940

tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 9000

cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 9060

tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 9120

agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 9180

ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 9240

ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 9300

aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 9360

gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 9420

tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 9480

ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc 9540

cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9600

atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9660

cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9720

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9780

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9840

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9900

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9960

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 10020

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 10080

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 10140

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 10200

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 10260

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 10320

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 10380

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 10440

ggttcctggc cttttgctgg ccttttgctc acatgt 10476

<210> 3

<211> 3120

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgccaagctt gcatgcaggc ctctgcagtc gacgggcccg ggatccgatg 2280

ataaacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 2340

tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 2400

gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 2460

ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 2520

gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 2580

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc 2640

tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccataga tctagatgca 2700

ttcgcgaggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 2760

accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 2820

atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 2880

ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 2940

gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 3000

cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3060

tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3120

<210> 4

<211> 14138

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

ggcgcgccct ctacctgctc tcggacccgt gggggtgggg ggtggaggaa ggagtggggg 60

gtcggtcctg ctggcttgtg ggtgggaggc gcatgttctc caaaaacccg cgcgagctgc 120

aatcctgagg gagctgcagt ggaggaggcg gagagaaggc cgcacccttc tccgcagggg 180

gaggggagtg ccgcaatacc tttatgggag ttctctgctg cctccttttc ctaaggaccg 240

ccctgggcct agaaaaatcc ctccctcccc cgcgatctcg tcatcgcctc catgtcagtt 300

tgctccttct cgattatggg cgggattctt ttgccctggc gcgccccaga cccgggcctg 360

gggggcaagt cggggggcgg ggggaggtcg ggcagggtcc cctgggagga tggggacgtg 420

ctgtgcccct agcggccacc agagggcacc aggacaccac tgcggtcggc tcagcggctc 480

ctgccctggt cagggggcgc caggtcctgc ccctcctggg gagggcgggg ggcgagaagg 540

gcgattttaa ttaacccacg tttcaacatg cacatcccag taatttggaa acattttgtt 600

tccaaagatt cacttaacat tggtttagca acatgaagct ttctatgcaa cccaaggact 660

cagtttttgg cctgttttag tgacaggcaa tcagcaacat gctgcatttc tctccagtgt 720

tgtaatcaaa gaaaccctcc catagcttta aatgatattc cttccccttc caattatgtg 780

gggggaaaac aaccctattc tccacccaga agtgttaact caagaattac attttcaaga 840

agtttccaga ttcgtaaaac cagaattaga tgtctttcac ctaaatgtct cggtgttgac 900

caaaggaaca cacaggtttc tcatttaact tttttaatgg gtctcaaaat tctgtgacaa 960

atttttggtc aagttgtttc cattaaaaag tactgatttt aaaaactaat aacttaaaac 1020

tgccacacgc aaaaaagaaa accaaagtgg tccacaaaac attctccttt ccttctgaag 1080

gttttacgat gcattgttat cattaaccag tcttttacta ctaaacttaa atggccaatt 1140

gaaacaaaca gttctgagac cgttcttcca ccactgatta agagtggggt ggcaggtatt 1200

agggataatg ctagcttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt 1260

cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga 1320

ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt 1380

ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa 1440

gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta 1500

gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag 1560

ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt 1620

gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt 1680

gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt 1740

cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt 1800

cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg 1860

gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc 1920

gcccttgctc accatggtgg cgtcgaccgt acgtcacgac acctgaaatg gaagaaaaaa 1980

actttgaacc actgtctgag gcttgagaat gaaccaagat ccaaactcaa aaagggcaaa 2040

ttccaaggag aattacatca agtgccaagc tggcctaact tcagtctcca cccactcagt 2100

gtggggaaac tccatcgcat aaaacccctc cccccaacct aaagacgacg tactccaaaa 2160

gctcgagaac taatcgaggt gcctggacgg cgcccggtac tccgtggagt cacatgaagc 2220

gacggctgag gacggaaagg cccttttcct ttgtgtgggt gactcacccg cccgctctcc 2280

cgagcgccgc gtcctccatt ttgagctccc tgcagcaggg ccgggaagcg gccatctttc 2340

cgctcacgca actggtgccg accgggccag ccttgccgcc cagggcgggg cgatacacgg 2400

cggcgcgagg ccaggcacca gagcaggccg gccagcttga gactaccccc gtccgattct 2460

cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg cgctgggacg cacgggcccc 2520

gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc agatcttggc ccgcatttac 2580

aagactatct tgccagaaaa aaagcgtcgc agcaggtcat caaaaatttt aaatggctag 2640

agacttatcg aaagcagcga gacaggcgcg aaggtgccac cagattcgca cgcggcggcc 2700

ccagcgccca ggccaggcct caactcaagc acgaggcgaa ggggctcctt aagcgcaagg 2760

cctcgaactc tcccacccac ttccaacccg aagctcggga tcaagaatca cgtactgcag 2820

ccagtggaag taattcaagg cacgcaaggg ccataacccg taaagaggcc aggcccgcgg 2880

gaaccacaca cggcacttac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc 2940

acggcgacta ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag 3000

tacacgacat cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg 3060

ccgacccctc cccccaactt ctcggggact gtgggcgatg tgcgctctgc ccactgacgg 3120

gcaccggagc cctagattcg attccctttg gggcaaaact caccgcctaa tcccctataa 3180

ctctaccggg gagcccggtg gagagcagac gggctgacgc tgccacctgc cggccatccc 3240

aggataggac cgccgtattc aagtcgccct caggaaggac cctcggggca ccagaggcct 3300

tcgaagcccc aatgagtgag gcaactgagg gtcgcgggtg ccattacaag gcccagccaa 3360

ggcctagagc caaggcttga accgtggggg acccccaagc cccacctgcc caggaacagc 3420

agacactggg acactttgtt tcaggtcctg cccaggcccc tcccactgtg aggctgggat 3480

ttgtcgccca gggtgcagat gagaagagtg gggaaagcag tcctgagcca ggaaattcta 3540

ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct 3600

gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag 3660

gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt 3720

caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc 3780

acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct 3840

ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag 3900

gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc 3960

ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc 4020

ttcctcatct ccgggccttt cgacctccta gggccaccat ggtgagcaag ggcgaggacg 4080

acaacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga 4140

acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga 4200

ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc 4260

ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact 4320

tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg 4380

gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga 4440

agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct 4500

gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc 4560

agaggctgaa gctgaaggac ggcggccact acgacgccga ggtcaagacc acctacaagg 4620

ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagctg gacatcacct 4680

cccacaacga ggactacacc atcgtggaac agtacgagcg cgccgagggc cgccactcca 4740

ccggcggcat ggacgagctg tacaagtgag gatccgctga tcagcctcga ctgtgccttc 4800

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 4860

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4920

tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 4980

tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaacccttct 5040

gaggcggaaa gaaccagctg ccttaatata acttcgtata atgtatgcta tacgaagtta 5100

ttaggtctga agaggagttt acgtccagcc aattctgtgg aatgtgtgtc agttagggtg 5160

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 5220

agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 5280

tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 5340

gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 5400

cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 5460

aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggcggcc gcgccaccat 5520

gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg 5580

caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg 5640

ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat 5700

cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag 5760

cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc 5820

ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc 5880

cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag 5940

cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga 6000

gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga 6060

cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaga 6120

attcgcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 6180

gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 6240

gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac 6300

ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 6360

aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 6420

catgtctgta taccgctcga ctagagcttg cggaaccctt aatataactt cgtataatgt 6480

atgctatacg aagttattag gtccgctggc catctacgag ccaaagactt tcaaatcttt 6540

ggctgccttg gccagtagga ggcgacacga aggatttgct gctgccttgg gggatgggaa 6600

ggaacctgaa ggcatttttt ccagagtggt gcagtaccac tgaggactgt tgctgtattg 6660

attaggaaaa gagacagagt aatttgcagt ttgtttgatt tatactgggc tgcaggtcga 6720

gggatcttca taagagaaga gggacagcta tgactgggag tagtcaggag aggaggaaaa 6780

atctggctag taaaacatgt aaggaaaatt ttagggatgt taaagaaaaa aataacacaa 6840

aacaaaatat aaaaaaaatc taacctcaag tcaaggcttt tctatggaat aaggaatgga 6900

cagcaggggg ctgtttcata tactgatgac ctctttatag ccacctttgt tcatggcagc 6960

cagcatatgg catatgttgc caaactctaa accaaatact cattctgatg ttttaaatga 7020

tttgccctcc catatgtcct tccgagtgag agacacaaaa aattccaaca cactattgca 7080

atgaaaataa atttccttta ttagccagaa gtcagatgct caaggggctt catgatgtcc 7140

ccataatttt tggcagaggg aaaaagatct cagtggtatt tgtgagccag ggcattggcc 7200

acaccagcca ccaccttctg ataggcagcc tgcggtacct tacatggtgg cgaattcgtt 7260

tgccaaaatg atgagacagc acaataacca gcacgttgcc caggagctgt aggaaaaaga 7320

agaaggcatg aacatggtta gcagaggctc tagagccgcc ggtcacacgc cagaagccga 7380

accccgccct gccccgtccc ccccgaaggc agccgtcccc ctgcggcagc cccgaggctg 7440

gagatggaga aggggacggc ggcgcggcga cgcacgaagg ccctccccgc ccatttcctt 7500

cctgccggcg ccgcaccgct tcgcccgcgc ccgctagagg gggtgcggcg gcgcctccca 7560

gatttcggct ccgccagatt tgggacaaag gaagtccctg cgccctctcg cacgattacc 7620

ataaaaggca atggctgcgg ctcgccgcgc ctcgacagcc gccggcgctc cggggccgcc 7680

gcgcccctcc cccgagccct ccccggcccg aggcggcccc gccccgcccg gcacccccac 7740

ctgccgccac cccccgcccg gcacggcgag ccccgcgcca cgccccgcac ggagccccgc 7800

acccgaagcc gggccgtgct cagcaactcg gggagggggg tgcagggggg ggttacagcc 7860

cgaccgccgc gcccacaccc cctgctcacc cccccacgca cacaccccgc acgcagcctt 7920

tgttcccctc gcagcccccc cgcaccgcgg ggcaccgccc ccggccgcgc tcccctcgcg 7980

cacacgcgga gcgcacaaag ccccgcgccg cgcccgcagc gctcacagcc gccgggcagc 8040

gcgggccgca cgcggcgctc cccacgcaca cacacacgca cgcacccccc gagccgctcc 8100

cccccgcaca aagggccctc ccggagccct ttaaggcttt cacgcagcca cagaaaagaa 8160

acgagccgtc attaaaccaa gcgctaatta cagcccggag gagaagggcc gtcccgcccg 8220

ctcacctgtg ggagtaacgc ggtcagtcag agccggggcg ggcggcgcga ggcggcgcgg 8280

agcggggcac ggggcgaagg caacgcagcg actcccgccc gccgcgcgct tcgcttttta 8340

tagggccgcc gccgccgccg cctcgccata aaaggaaact ttcggagcgc gccgctctga 8400

ttggctgccg ccgcacctct ccgcctcgcc ccgccccgcc cctcgccccg ccccgccccg 8460

cctggcgcgc gccccccccc cccccgcccc catcgctgca caaaataatt aaaaaataaa 8520

taaatacaaa attgggggtg gggagggggg ggagatgggg agagtgaagc agaacgtggg 8580

gctcacctcg acccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa 8640

agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt 8700

caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc 8760

gtaaatagtc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat 8820

acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta 8880

ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg 8940

attactatta ataactagtc aataatcaat gtcgtaaatg tcgtaaatgt ctcagctagt 9000

caggtagtaa aaggtgtcaa ctaggcagtg gcagagcagg attcaaattc agggctgttg 9060

tgatgcctcc gcagactctg agcgccacct ggtggtaatt tgtctgtgcc tcttctgacg 9120

tggaagaaca gcaactaaca cactaacacg gcatttacta tgggccagcc attgtacgcg 9180

ttgcttaacc tgattcttgg gcgttgtcct gcaggggatt gagcaggtgt acgaggacga 9240

gcccaatttc tctatattcc cacagtcttg agtttgtgtc acaaaataat tatagtgggg 9300

tggagatggg aaatgagtcc aggcaacacc taagcctgat tttatgcatt gagactgcgt 9360

gttattacta aagatctttg tgtcgcaatt tcctgatgaa gggagatagg ttaaaaagca 9420

cggatctact gagttttaca gtcatcccat ttgtagactt ttgctacacc accaaagtat 9480

agcatctgag attaaatatt aatctccaaa ccttaggccc cctcacttgc atccttacgg 9540

tcagataact ctcactcata ctttaagccc attttgtttg ttgtacttgc tcatccagtc 9600

ccagacatag cattggcttt ctcctcacct gttttaggta gccagcaagt catgaaatca 9660

gataagttcc accaccaatt aacactaccc atcttgagca taggcccaac agtgcattta 9720

ttcctcattt actgatgttc gtgaatattt accttgattt tcattttttt ctttttctta 9780

agctgggatt ttactcctga ccctattcac agtcagatga tcttgactac cactgcgatt 9840

ggacctgagg ttcagcaata ctccccttta tgtcttttga atacttttca ataaatctgt 9900

ttgtattttc attagttagt aactgagctc agttgccgta atgctaatag cttccaaact 9960

agtgtctctg tctccagtat ctgataaatc ttaggtgttg ctgggacagt tgtcctaaaa 10020

ttaagataaa gcatgaaaat aactgacaca actccattac tggctcctaa ctacttaaac 10080

aatgcattct atcatcacaa atgtgaaaaa ggagttccct cagtggacta accttatctt 10140

ttctcaacac ctttttcttt gcacaatttt ccacacatgc ctacaaaaag tacttatgcg 10200

gccgccataa aagttttgtt actttataga agaaattttg agtttttgtt ttttttaata 10260

aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat 10320

aataaaactt aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac 10380

acatgcgtca attttacaca tgattatctt taacgtacgt cacaatatga ttatctttct 10440

agggttaatc tagctgcgtg ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc 10500

tgtaaaacac atttgcaccg cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga 10560

acgaggcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 10620

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 10680

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct 10740

gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 10800

ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 10860

gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 10920

ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 10980

gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 11040

tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 11100

tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 11160

ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac 11220

ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 11280

ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 11340

cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 11400

ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 11460

tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 11520

cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 11580

actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 11640

aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 11700

tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 11760

ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 11820

tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 11880

gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 11940

gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 12000

tattgctgat aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg 12060

gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 12120

ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 12180

gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 12240

aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 12300

ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 12360

ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 12420

tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 12480

gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 12540

agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 12600

taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 12660

gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 12720

gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 12780

caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 12840

aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 12900

tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 12960

acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 13020

ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 13080

gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc 13140

tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 13200

agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc 13260

tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 13320

cacaggaaac agctatgacc atgattacgc caagcgcgcc cgccgggtaa ctcacggggt 13380

atccatgtcc atttctgcgg catccagcca ggatacccgt cctcgctgac gtaatatccc 13440

agcgccgcac cgctgtcatt aatctgcaca ccggcacggc agttccggct gtcgccggta 13500

ttgttcgggt tgctgatgcg cttcgggctg accatccgga actgtgtccg gaaaagccgc 13560

gacgaactgg tatcccaggt ggcctgaacg aacagttcac cgttaaaggc gtgcatggcc 13620

acaccttccc gaatcatcat ggtaaacgtg cgttttcgct caacgtcaat gcagcagcag 13680

tcatcctcgg caaactcttt ccatgccgct tcaacctcgc gggaaaaggc acgggcttct 13740

tcctccccga tgcccagata gcgccagctt gggcgatgac tgagccggaa aaaagacccg 13800

acgatatgat cctgatgcag ctagattaac cctagaaaga tagtctgcgt aaaattgacg 13860

catgcattct tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt 13920

taggacatct cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg 13980

tcactgattt tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg 14040

tgacttttaa gatttaactc atacgataat tatattgtta tttcatgttc tacttacgtg 14100

ataacttatt atatatatat tttcttgtta tagatatc 14138

<210> 5

<211> 345

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

ggcgcgccct ctacctgctc tcggacccgt gggggtgggg ggtggaggaa ggagtggggg 60

gtcggtcctg ctggcttgtg ggtgggaggc gcatgttctc caaaaacccg cgcgagctgc 120

aatcctgagg gagctgcagt ggaggaggcg gagagaaggc cgcacccttc tccgcagggg 180

gaggggagtg ccgcaatacc tttatgggag ttctctgctg cctccttttc ctaaggaccg 240

ccctgggcct agaaaaatcc ctccctcccc cgcgatctcg tcatcgcctc catgtcagtt 300

tgctccttct cgattatggg cgggattctt ttgccctggc gcgcc 345

<210> 6

<211> 1012

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

cttaacctga ttcttgggcg ttgtcctgca ggggattgag caggtgtacg aggacgagcc 60

caatttctct atattcccac agtcttgagt ttgtgtcaca aaataattat agtggggtgg 120

agatgggaaa tgagtccagg caacacctaa gcctgatttt atgcattgag actgcgtgtt 180

attactaaag atctttgtgt cgcaatttcc tgatgaaggg agataggtta aaaagcacgg 240

atctactgag ttttacagtc atcccatttg tagacttttg ctacaccacc aaagtatagc 300

atctgagatt aaatattaat ctccaaacct taggccccct cacttgcatc cttacggtca 360

gataactctc actcatactt taagcccatt ttgtttgttg tacttgctca tccagtccca 420

gacatagcat tggctttctc ctcacctgtt ttaggtagcc agcaagtcat gaaatcagat 480

aagttccacc accaattaac actacccatc ttgagcatag gcccaacagt gcatttattc 540

ctcatttact gatgttcgtg aatatttacc ttgattttca tttttttctt tttcttaagc 600

tgggatttta ctcctgaccc tattcacagt cagatgatct tgactaccac tgcgattgga 660

cctgaggttc agcaatactc ccctttatgt cttttgaata cttttcaata aatctgtttg 720

tattttcatt agttagtaac tgagctcagt tgccgtaatg ctaatagctt ccaaactagt 780

gtctctgtct ccagtatctg ataaatctta ggtgttgctg ggacagttgt cctaaaatta 840

agataaagca tgaaaataac tgacacaact ccattactgg ctcctaacta cttaaacaat 900

gcattctatc atcacaaatg tgaaaaagga gttccctcag tggactaacc ttatcttttc 960

tcaacacctt tttctttgca caattttcca cacatgccta caaaaagtac tt 1012

<210> 7

<211> 1073

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

gtgctgagtc cttttcccat cccacccacc tggagctccc ctcttccagt cctgagccac 60

ttgaactggc ctggtttttg ccatcctgcg ctgccctctc tccggactcg agccactgct 120

gagggcctca ggccagtcca tcctcgtctt gtctctttcg ccctgctctt tccccacctt 180

gagcgctctt aaccagcctg gcccgtgcca cctctactct gccatcgaat gctgccccac 240

tttctcgagt ccgccacttc tcccagcttc accggtaccc actgtttccc ctagtccagg 300

caggtaccac tttccctgag cgtcctcctc ctctctcctg ggcctgtgct gcttcttttc 360

ccgctctctg gcctgggccg tttcttcggc cagcccccga gccttccatg ccctttcctt 420

caggtttctg ctcttcatcc ttggtctctg ccatctgttg ccatgtaagg gtgctctttc 480

ctgagccatc gccctcaagg cgctctgctc ctcaagtgga tgcttccctc gcctggctca 540

cctcctgctc tctctcctgc ccccttcacc tgcgtgccct cctcattctc cctctgtgcc 600

acctctggcc ttgcactgta ggctctctct tggggatgtt tctccttctc cacacacttc 660

tctttcactc tgtcctcttg ctttgtgtgg gcctgcagcg ttaccctttt ttctgggcac 720

actcagagca ccctcctctt tctggttctg ggccacctgt ctgtcctcgg gtcatcttgc 780

tctctctgcc tggatgccct cctgtggctt tgggcagctt ctccctcctt cagagtgcac 840

cgccagttct cctaggcccg gtcacttccc cttcccaggg gacctagagc cctgctaggt 900

cctctctctc cacaacctgg gcccccaaac ctttccaaaa caccttgctt tctgcctcca 960

ttggtcttgt gttccagagc cagagtcact atatgtccca gaaccaggat tccctctggt 1020

tctgagggct tttatcgcat cccctgcctg gctgcagtgg gtctttgggc gcc 1073

<210> 8

<211> 260

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

gacaggccac agaagagcct ctactcctcc ctctgtcccc gaggctgtct ccctcccagt 60

cttcccagct caggccagtc cccaggcctc tcttccctgc cagagcccgt caggttcggt 120

tactttgggg cccagagagg accctgtgaa ggaagcgtgg gtaggggcac gggaatgggg 180

aggatgcctg aagaggcccc cttagccaga agaggagcag aagaggagca ggtacccaga 240

agaggagcag ttcagggaaa 260

<210> 9

<211> 546

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

aaatacccac gtttattggg acaaaagttg ttagggaaaa tggggcctca gagttatgat 60

tcaagtcata attctttcca tttataattt cactcgagac tctgttaact gattccttgt 120

gtgttgtatc ttactcctca gctcacaatt acttttagtt attcacctta actgtatgaa 180

taacagtgga gaaaaggatt ctaccagaat actctaatta tggttttgag tcccctttcc 240

agactgaaga tttttcagtc tttttgatct gaggtgattt ttcagtcttt tcgatctgag 300

gtgacagtct caagctcctc aattcaccca gtctcttgat acttgtccat ttagggccac 360

caaagctact ttgacttcat actagagagt caattaatga ggccattctc tgatggacag 420

gtgaagcagg caaggtgact atattttgac taaacggtag aaaacagcct gagtgttaac 480

agtgtagcct ataaaaccca gagctgccca ccctgatcta aacttccagg aacataagaa 540

cgcgcc 546

<210> 10

<211> 1009

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

agtaggtcac atttcagtaa aacctggctt tgtggattga gcatggtctg tctcttcctg 60

gtacttcatt agtcccctaa gtgggatttg ctgagcaaga ctcctcaatt acagaaatac 120

tccagtttag aattctcgca aaggcttttt gtttccacaa gtagaatcta gaaagcaatc 180

tcaagtaaca acagcagaga cctgaatccc aatccatctt tcctgtgtgt cctcttttac 240

ctccttccct ttcatgttga accaacagtc ctttttcagt ctagaagcta gtacgaaaga 300

aatgtacaga tgtaggtacc aagcaaagcc attagccaat aactggtgag atggagctaa 360

gaggaaataa aagtgttcct aagaatagca cagcagaagc tagatccaca gatcttaaaa 420

caattttggt tgagtaagag tagaggcaaa agaggaagct aataatgcag tttttaggag 480

ctaagagcca gataaagggt aagggcagga ggaagtgcta tctcagctaa cgagatacat 540

gaaacaacgg tggaagtcca gcaggcacaa gatgagttga gaagcaatca gggccagaag 600

gatgtgcaag gcctcaaaat aaaaaagcac agggccacag ggaaccttat ggaaattaaa 660

aggaagagga tgcagtcagg agaggaaaaa atagtgctcc ctcccccatg cccaaggaag 720

cagctgagca gccagtactt gggaagttag tagtaataag ttggtaagag ggagttctgt 780

tcgtggctca atggttaaca aatcagacta gaaaccgtga ggttgcgggt ttgatccctg 840

gccttgctca gtgggttaag gatccggcat tgccgtgacc tgtggtgtag gtcacagacg 900

tggctcagtt cccgcattcc tgtggctctg gtgtaggctg gtggctacag ctctgattag 960

acccctaggc tgggaacctc catatgccct ggaagtggcc gtagaaaag 1009

<210> 11

<211> 878

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

ggatggggac tcatgtgaat tttctaaagg tgctatttaa acggggggca cgagtgccgg 60

ctttggacag ggccgctcgc tctccaccct ttcttcttcc ccctcggccg cctctcaccc 120

cctgaggcct ctctcccccc acgacctcct ctctctcctc tgaaaccctc tcctcctcag 180

ctgcatccca ccctcgtggc ctctctctct ctctgtctgt cctgtgtcct ctctcactgg 240

gtttcagagc acagatgccc aaagcacaaa agcagttttc ccctggggtg ggaggaagca 300

agagactttg tacctatttt gtatgtgtat aataatttga gatgttttta attattttga 360

ttgctggaat aaagcatgtg gaaatgaccc aaaccaatct tgcactggcc tcctgatttc 420

cttccttgga gacggaggga gggggagacc tgggggaggg cgcttggggg ggggtgggct 480

ctcttctttc tgcgctcccc ccccccacct ccaacacctt gacgacccct cctgcttccg 540

cttgcctttc tcaggcttta acactttctc ctcgccctct cagcatgcgc atgcgcgtgc 600

ctctacctcc cccgcacatc ctggcctgcc caccctgaat ggcctggccc agcgatgcca 660

ccaactctct cgctccgtcc acggctgggg aggggggcac tctgcagggt tggggggcac 720

tgggaggctg ggttgggtga gggaggggtg cctgggcccc caccccccag caagttctct 780

ccctaggcga actggagggt cgtctggcct cttgagcctt gttgctggct ctgagctcta 840

ccaagagagt gaccagcagg accgcaccat cacgcgcc 878

<210> 12

<211> 727

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

gtggttgctg agactgcgtg ggggcccaag gagacctgga gaaaggaatg cttcctgctc 60

cttcttctgg ggccccagga gagccttccc agggccttgg agaggtgctg tccagggact 120

aaccctgtgc tctaggaagg ctgcaggccc tgaccagctg ggcaggtcct gggtccctcc 180

tggccttcta agttccccaa acatgagacc tctgggtgtg gggtggcctg gggaggtcat 240

tttgcccagg ccctacctcc tgcccattcc taaccctttt taaaaatctg tgcgtcctct 300

tcttccttct tctccctccc ttcccttttc gctcaccctc tgctgctggc ctgagagccg 360

gaggccccca gggggaaggc gactggtctc ctccccagtc tcagggaagg gagacagaga 420

atccaggaag ccagaactca gcagacgaag cacccaggga cctagagatg ggttgaaaag 480

ttgacagctg tcccacctgc ctcccaaggt ctcagggcct aaacctccaa ggcaggaaag 540

gcccctgtcc ctccctgggg tccatagaaa gagggacaag tctgcacgga ccatttgctg 600

taatattaac accttggctg tcattaggta gtcttggctg ttaattatgt cctgtgataa 660

tgtattatta gcacgccgac cacatagggt agggaactgc agctagtaaa caaaagtttg 720

ttcctat 727

<210> 13

<211> 10339

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

ggatggggac tcatgtgaat tttctaaagg tgctatttaa acggggggca cgagtgccgg 60

ctttggacag ggccgctcgc tctccaccct ttcttcttcc ccctcggccg cctctcaccc 120

cctgaggcct ctctcccccc acgacctcct ctctctcctc tgaaaccctc tcctcctcag 180

ctgcatccca ccctcgtggc ctctctctct ctctgtctgt cctgtgtcct ctctcactgg 240

gtttcagagc acagatgccc aaagcacaaa agcagttttc ccctggggtg ggaggaagca 300

agagactttg tacctatttt gtatgtgtat aataatttga gatgttttta attattttga 360

ttgctggaat aaagcatgtg gaaatgaccc aaaccaatct tgcactggcc tcctgatttc 420

cttccttgga gacggaggga gggggagacc tgggggaggg cgcttggggg ggggtgggct 480

ctcttctttc tgcgctcccc ccccccacct ccaacacctt gacgacccct cctgcttccg 540

cttgcctttc tcaggcttta acactttctc ctcgccctct cagcatgcgc atgcgcgtgc 600

ctctacctcc cccgcacatc ctggcctgcc caccctgaat ggcctggccc agcgatgcca 660

ccaactctct cgctccgtcc acggctgggg aggggggcac tctgcagggt tggggggcac 720

tgggaggctg ggttgggtga gggaggggtg cctgggcccc caccccccag caagttctct 780

ccctaggcga actggagggt cgtctggcct cttgagcctt gttgctggct ctgagctcta 840

ccaagagagt gaccagcagg accgcaccat cacgcgcccc agacccgggc ctggggggca 900

agtcgggggg cggggggagg tcgggcaggg tcccctggga ggatggggac gtgctgtgcc 960

cctagcggcc accagagggc accaggacac cactgcggtc ggctcagcgg ctcctgccct 1020

ggtcaggggg cgccaggtcc tgcccctcct ggggagggcg gggggcgaga agggcgattg 1080

cagaaatggt tgaactcccg agagtgtcct acacctaggg gagaagcagc caaggggttg 1140

tttcccacca aggacgaccc gtctgcgcac aaacggatga gcccatcaga caaagacata 1200

ttcattctct gctgcaaact tggcatagct ctgctttgcc tggggctatt gggggaagtt 1260

gcggttcgtg ctcgcagggc tctcaccctt gactctttca ataataactc ttctgtgcaa 1320

gattacaatc taaacaattc ggagaactcg accttcctcc tgaggcaagg accacagcca 1380

acttcctctt acaagccgca tcgattttgt ccttcagaaa tagaaataag aatgcttgct 1440

aaaaattata tttttaccaa taagaccaat ccaataggta gattattagt tactatgtta 1500

agaaatgaat cattatcttt tagtactatt tttactcaaa ttcagaagtt agaaatggga 1560

atagaaaata gaaagagacg ctcaacctca attgaagaac aggtgcaagg actattgacc 1620

acaggcctag aagtaaaaaa gggaaaaaag agtgtttttg tcaaaatagg agacaggtgg 1680

tggcaaccag ggacttatag gggaccttac atctacagac caacagatgc ccccttacca 1740

tatacaggaa gatatgactt aaattgggat aggtgggtta cagtcaatgg ctataaagtg 1800

ttatatagat ccctcccctt tcgtgaaaga ctcgccagag ctagacctcc ttggtgtatg 1860

ttgtctcaag aaaagaaaga cgacatgaaa caacaggtac atgattatat ttatctagga 1920

acaggaatgc acttttgggg aaagattttc cataccaagg aggggacagt ggctggacta 1980

atagaacatt attctgcaaa aacttatggc atgagttatt atgattagcc ttgatttgcc 2040

caaccttgcg gttcccaagg cttaagtaag tttttggtta caaactgttc ttaaaacaag 2100

gatgtgagac aagtggtttc ctgacttggt ttggtatcaa aggttctgat ctgagctctg 2160

agtgttctat tttcctatgt tcttttggaa tttatccaaa tcttatgtaa atgcttatgt 2220

aaaccaagat ataaaagagt gctgattttt tgagtaaact tgcaacagtc ctaacattca 2280

cctcttgtgt gtttgtgtct gttcgccatc ccgtctccgc tcgtcactta tccttcactt 2340

tccagagggt ccccccgcag accccggcga ccctcaggtc ggccgactgc ggcatctaga 2400

gccaccatgg atagagttct gagcagagct gacaaagaaa ggctgctaga acttctaaaa 2460

cttcccagac aactatgggg ggattttgga agaatgcagc aggcatataa gcagcagtca 2520

ctgctactgc acccagacaa aggtggaagc catgccttaa tgcaggaatt gaacagtctc 2580

tggggaacat ttaaaactga agtatacaat ctgagaatga atctaggagg aaccggcttc 2640

caggtaagaa ggctacatgc ggatgggtgg aatctaagta ccaaagacac ctttggtgat 2700

agatactacc agcggttctg cagaatgcct cttacctgcc tagtaaatgt taaatacagc 2760

tcatgtagtt gtatattatg cctgcttaga aagcaacata gagagctcaa agacaaatgt 2820

gatgccaggt gcctagtact tggagaatgt ttttgtcttg aatgttacat gcaatggttt 2880

ggaacaccaa cccgagatgt gctgaacctg tatgcagact tcattgcaag catgcctata 2940

gactggctgg acctggatgt gcacagcgtg tataatccaa aacggcggag cgaggaactg 3000

aggagagcgg ccacagtcca ctacacgatg actactggtc attcagctat ggaagcaagt 3060

acttcacaag ggaatggaat gatttcttca gaaagtggga ccccagctac cagtcgccgc 3120

ctaagactgc cgagtcttct gagcaacccg acctattctg ttatgaggag ccactcctat 3180

cccccaaccc gagttctcca acagatacac ccgcacatac tgctggaaga agacgaaatc 3240

cttgtgttgc tgagcccgat gacagcatat ccccggaccc ccccagaact cctgtatcca 3300

gaaagcgacc aagaccagct ggagccactg gaggaggagg aggaggagta catgccaatg 3360

gaggatctgt atttggacat cctaccgggg gaacaagtac cccagctcat ccccccccct 3420

atcattccca gggcgggtct gagtccatgg gagggtctga ttcttcggga tttgcagagg 3480

gctcatttcg atccgatcct agatgcgagt cagagaatga gagctactca cagagctgct 3540

ctcagagctc attcaatgca acgccaccta agaaggctag ggaggaccct gctcctagtg 3600

actttcctag cagccttact gggtatttgt ctcatgctat ttattctaat aaaacgttcc 3660

cggcatttct agggccgcga ctctagagtc ggggcggccg gccgcttcga gcagacatga 3720

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3780

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3840

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3900

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggaacaa caacaattgc 3960

attcatttta tgtttcaggt tcagggggag gtgtgggagg tctgaggcgg aaagaaccag 4020

ctgccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc tgaagaggag 4080

tttacgtcca gccaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 4140

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 4200

agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 4260

ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 4320

ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 4380

ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 4440

tcccgggagc ttgtatatcc attttcggcg gccgcgccac catgaccgag tacaagccca 4500

cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 4560

tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 4620

ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 4680

cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 4740

tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 4800

aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 4860

ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 4920

gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 4980

acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 5040

gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agaattcgcg ggactctggg 5100

gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc 5160

cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 5220

ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 5280

taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 5340

gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgct 5400

cgactagagc ttgcggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat 5460

taggtccgct ggccatctac gagccaaaga ctttcaaatc tttggctgcc ttggccagta 5520

ggaggcgaca cgaaggattt gctgctgcct tgggggatgg gaaggaacct gaaggcattt 5580

tttccagagt ggtgcagtac cactgaggac tgttgctgta ttgattagga aaagagacag 5640

agtaatttgc agtttgtttg atttatactg tggttgctga gactgcgtgg gggcccaagg 5700

agacctggag aaaggaatgc ttcctgctcc ttcttctggg gccccaggag agccttccca 5760

gggccttgga gaggtgctgt ccagggacta accctgtgct ctaggaaggc tgcaggccct 5820

gaccagctgg gcaggtcctg ggtccctcct ggccttctaa gttccccaaa catgagacct 5880

ctgggtgtgg ggtggcctgg ggaggtcatt ttgcccaggc cctacctcct gcccattcct 5940

aacccttttt aaaaatctgt gcgtcctctt cttccttctt ctccctccct tcccttttcg 6000

ctcaccctct gctgctggcc tgagagccgg aggcccccag ggggaaggcg actggtctcc 6060

tccccagtct cagggaaggg agacagagaa tccaggaagc cagaactcag cagacgaagc 6120

acccagggac ctagagatgg gttgaaaagt tgacagctgt cccacctgcc tcccaaggtc 6180

tcagggccta aacctccaag gcaggaaagg cccctgtccc tccctggggt ccatagaaag 6240

agggacaagt ctgcacggac catttgctgt aatattaaca ccttggctgt cattaggtag 6300

tcttggctgt taattatgtc ctgtgataat gtattattag cacgccgacc acatagggta 6360

gggaactgca gctagtaaac aaaagtttgt tcctatatgc ggccgccata aaagttttgt 6420

tactttatag aagaaatttt gagtttttgt tttttttaat aaataaataa acataaataa 6480

attgtttgtt gaatttatta ttagtatgta agtgtaaata taataaaact taatatctat 6540

tcaaattaat aaataaacct cgatatacag accgataaaa cacatgcgtc aattttacac 6600

atgattatct ttaacgtacg tcacaatatg attatctttc tagggttaat ctagctgcgt 6660

gttctgcagc gtgtcgagca tcttcatctg ctccatcacg ctgtaaaaca catttgcacc 6720

gcgagtctgc ccgtcctcca cgggttcaaa aacgtgaatg aacgaggcgc gctcactggc 6780

cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 6840

agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 6900

ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 6960

ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 7020

tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 7080

aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 7140

acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 7200

tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 7260

caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 7320

gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 7380

tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 7440

taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 7500

tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 7560

gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 7620

gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 7680

cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 7740

tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 7800

tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 7860

atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 7920

ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 7980

gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 8040

gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 8100

gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 8160

gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 8220

gccggtgagc gtggttcacg cggtatcatt gcagcactgg ggccagatgg taagccctcc 8280

cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 8340

atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 8400

tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 8460

ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 8520

gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 8580

tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 8640

ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 8700

ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 8760

gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 8820

ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 8880

tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 8940

ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 9000

agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 9060

agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 9120

gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 9180

tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 9240

accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 9300

gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 9360

attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 9420

gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 9480

gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 9540

catgattacg ccaagcgcgc ccgccgggta actcacgggg tatccatgtc catttctgcg 9600

gcatccagcc aggatacccg tcctcgctga cgtaatatcc cagcgccgca ccgctgtcat 9660

taatctgcac accggcacgg cagttccggc tgtcgccggt attgttcggg ttgctgatgc 9720

gcttcgggct gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg 9780

tggcctgaac gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca 9840

tggtaaacgt gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt 9900

tccatgccgc ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat 9960

agcgccagct tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca 10020

gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt 10080

gctctctctt tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg 10140

cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata 10200

acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact 10260

catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata 10320

ttttcttgtt atagatatc 10339

<210> 14

<211> 421

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

Met Asp Arg Val Leu Ser Arg Ala Asp Lys Glu Arg Leu Leu Glu Leu

1 5 10 15

Leu Lys Leu Pro Arg Gln Leu Trp Gly Asp Phe Gly Arg Met Gln Gln

20 25 30

Ala Tyr Lys Gln Gln Ser Leu Leu Leu His Pro Asp Lys Gly Gly Ser

35 40 45

His Ala Leu Met Gln Glu Leu Asn Ser Leu Trp Gly Thr Phe Lys Thr

50 55 60

Glu Val Tyr Asn Leu Arg Met Asn Leu Gly Gly Thr Gly Phe Gln Val

65 70 75 80

Arg Arg Leu His Ala Asp Gly Trp Asn Leu Ser Thr Lys Asp Thr Phe

85 90 95

Gly Asp Arg Tyr Tyr Gln Arg Phe Cys Arg Met Pro Leu Thr Cys Leu

100 105 110

Val Asn Val Lys Tyr Ser Ser Cys Ser Cys Ile Leu Cys Leu Leu Arg

115 120 125

Lys Gln His Arg Glu Leu Lys Asp Lys Cys Asp Ala Arg Cys Leu Val

130 135 140

Leu Gly Glu Cys Phe Cys Leu Glu Cys Tyr Met Gln Trp Phe Gly Thr

145 150 155 160

Pro Thr Arg Asp Val Leu Asn Leu Tyr Ala Asp Phe Ile Ala Ser Met

165 170 175

Pro Ile Asp Trp Leu Asp Leu Asp Val His Ser Val Tyr Asn Pro Lys

180 185 190

Arg Arg Ser Glu Glu Leu Arg Arg Ala Ala Thr Val His Tyr Thr Met

195 200 205

Thr Thr Gly His Ser Ala Met Glu Ala Ser Thr Ser Gln Gly Asn Gly

210 215 220

Met Ile Ser Ser Glu Ser Gly Thr Pro Ala Thr Ser Arg Arg Leu Arg

225 230 235 240

Leu Pro Ser Leu Leu Ser Asn Pro Thr Tyr Ser Val Met Arg Ser His

245 250 255

Ser Tyr Pro Pro Thr Arg Val Leu Gln Gln Ile His Pro His Ile Leu

260 265 270

Leu Glu Glu Asp Glu Ile Leu Val Leu Leu Ser Pro Met Thr Ala Tyr

275 280 285

Pro Arg Thr Pro Pro Glu Leu Leu Tyr Pro Glu Ser Asp Gln Asp Gln

290 295 300

Leu Glu Pro Leu Glu Glu Glu Glu Glu Glu Tyr Met Pro Met Glu Asp

305 310 315 320

Leu Tyr Leu Asp Ile Leu Pro Gly Glu Gln Val Pro Gln Leu Ile Pro

325 330 335

Pro Pro Ile Ile Pro Arg Ala Gly Leu Ser Pro Trp Glu Gly Leu Ile

340 345 350

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

355 360 365

Gln Arg Met Arg Ala Thr His Arg Ala Ala Leu Arg Ala His Ser Met

370 375 380

Gln Arg His Leu Arg Arg Leu Gly Arg Thr Leu Leu Leu Val Thr Phe

385 390 395 400

Leu Ala Ala Leu Leu Gly Ile Cys Leu Met Leu Phe Ile Leu Ile Lys

405 410 415

Arg Ser Arg His Phe

420

<210> 15

<211> 7246

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

ggcctaactg gccgcagaaa tggttgaact cccgagagtg tcctacacct aggggagaag 60

cagccaaggg gttgtttccc accaaggacg acccgtctgc gcacaaacgg atgagcccat 120

cagacaaaga catattcatt ctctgctgca aacttggcat agctctgctt tgcctggggc 180

tattggggga agttgcggtt cgtgctcgca gggctctcac ccttgactct ttcaataata 240

actcttctgt gcaagattac aatctaaaca attcggagaa ctcgaccttc ctcctgaggc 300

aaggaccaca gccaacttcc tcttacaagc cgcatcgatt ttgtccttca gaaatagaaa 360

taagaatgct tgctaaaaat tatattttta ccaataagac caatccaata ggtagattat 420

tagttactat gttaagaaat gaatcattat cttttagtac tatttttact caaattcaga 480

agttagaaat gggaatagaa aatagaaaga gacgctcaac ctcaattgaa gaacaggtgc 540

aaggactatt gaccacaggc ctagaagtaa aaaagggaaa aaagagtgtt tttgtcaaaa 600

taggagacag gtggtggcaa ccagggactt ataggggacc ttacatctac agaccaacag 660

atgccccctt accatataca ggaagatatg acttaaattg ggataggtgg gttacagtca 720

atggctataa agtgttatat agatccctcc cctttcgtga aagactcgcc agagctagac 780

ctccttggtg tatgttgtct caagaaaaga aagacgacat gaaacaacag gtacatgatt 840

atatttatct aggaacagga atgcactttt ggggaaagat tttccatacc aaggagggga 900

cagtggctgg actaatagaa cattattctg caaaaactta tggcatgagt tattatgatt 960

agccttgatt tgcccaacct tgcggttccc aaggcttaag taagtttttg gttacaaact 1020

gttcttaaaa caaggatgtg agacaagtgg tttcctgact tggtttggta tcaaaggttc 1080

tgatctgagc tctgagtgtt ctattttcct atgttctttt ggaatttatc caaatcttat 1140

gtaaatgctt atgtaaacca agatataaaa gagtgctgat tttttgagta aacttgcaac 1200

agtcctaaca ttcacctctt gtgtgtttgt gtctgttcgc catcccgtct ccgctcgtca 1260

cttatccttc actttccaga gggtcccccc gcagaccccg gcgaccctca ggtcggccga 1320

ctgcggcagg cctcggcggc caagcttggc aatccggtac tgttggtaaa gccaccatgg 1380

aagatgccaa aaacattaag aagggcccag cgccattcta cccactcgaa gacgggaccg 1440

ccggcgagca gctgcacaaa gccatgaagc gctacgccct ggtgcccggc accatcgcct 1500

ttaccgacgc acatatcgag gtggacatta cctacgccga gtacttcgag atgagcgttc 1560

ggctggcaga agctatgaag cgctatgggc tgaatacaaa ccatcggatc gtggtgtgca 1620

gcgagaatag cttgcagttc ttcatgcccg tgttgggtgc cctgttcatc ggtgtggctg 1680

tggccccagc taacgacatc tacaacgagc gcgagctgct gaacagcatg ggcatcagcc 1740

agcccaccgt cgtattcgtg agcaagaaag ggctgcaaaa gatcctcaac gtgcaaaaga 1800

agctaccgat catacaaaag atcatcatca tggatagcaa gaccgactac cagggcttcc 1860

aaagcatgta caccttcgtg acttcccatt tgccacccgg cttcaacgag tacgacttcg 1920

tgcccgagag cttcgaccgg gacaaaacca tcgccctgat catgaacagt agtggcagta 1980

ccggattgcc caagggcgta gccctaccgc accgcaccgc ttgtgtccga ttcagtcatg 2040

cccgcgaccc catcttcggc aaccagatca tccccgacac cgctatcctc agcgtggtgc 2100

catttcacca cggcttcggc atgttcacca cgctgggcta cttgatctgc ggctttcggg 2160

tcgtgctcat gtaccgcttc gaggaggagc tattcttgcg cagcttgcaa gactataaga 2220

ttcaatctgc cctgctggtg cccacactat ttagcttctt cgctaagagc actctcatcg 2280

acaagtacga cctaagcaac ttgcacgaga tcgccagcgg cggggcgccg ctcagcaagg 2340

aggtaggtga ggccgtggcc aaacgcttcc acctaccagg catccgccag ggctacggcc 2400

tgacagaaac aaccagcgcc attctgatca cccccgaagg ggacgacaag cctggcgcag 2460

taggcaaggt ggtgcccttc ttcgaggcta aggtggtgga cttggacacc ggtaagacac 2520

tgggtgtgaa ccagcgcggc gagctgtgcg tccgtggccc catgatcatg agcggctacg 2580

ttaacaaccc cgaggctaca aacgctctca tcgacaagga cggctggctg cacagcggcg 2640

acatcgccta ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga 2700

tcaaatacaa gggctaccag gtagccccag ccgaactgga gagcatcctg ctgcaacacc 2760

ccaacatctt cgacgccggg gtcgccggcc tgcccgacga cgatgccggc gagctgcccg 2820

ccgcagtcgt cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc gtggactatg 2880

tggccagcca ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc gtggacgagg 2940

tgcctaaagg actgaccggc aagttggacg cccgcaagat ccgcgagatt ctcattaagg 3000

ccaagaaggg cggcaagatc gccgtgaatt ctcacggctt ccctcccgag gtggaggagc 3060

aggccgccgg caccctgccc atgagctgcg cccaggagag cggcatggat agacaccctg 3120

ctgcttgcgc cagcgccagg atcaacgtct aaggccgcga ctctagagtc ggggcggccg 3180

gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat 3240

gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 3300

tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 3360

gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga 3420

taaggatccg tttgcgtatt gggcgctctt ccgctgatct gcgcagcacc atggcctgaa 3480

ataacctctg aaagaggaac ttggttagct accttctgag gcggaaagaa ccagctgtgg 3540

aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 3600

agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 3660

agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 3720

cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 3780

ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 3840

ggaggctttt ttggaggcct aggcttttgc aaaaagctcg attcttctga cactagcgcc 3900

accatgaaga agcccgaact caccgctacc agcgttgaaa aatttctcat cgagaagttc 3960

gacagtgtga gcgacctgat gcagttgtcg gagggcgaag agagccgagc cttcagcttc 4020

gatgtcggcg gacgcggcta tgtactgcgg gtgaatagct gcgctgatgg cttctacaaa 4080

gaccgctacg tgtaccgcca cttcgccagc gctgcactac ccatccccga agtgttggac 4140

atcggcgagt tcagcgagag cctgacatac tgcatcagta gacgcgccca aggcgttact 4200

ctccaagacc tccccgaaac agagctgcct gctgtgttac agcctgtcgc cgaagctatg 4260

gatgctattg ccgccgccga cctcagtcaa accagcggct tcggcccatt cgggccccaa 4320

ggcatcggcc agtacacaac ctggcgggat ttcatttgcg ccattgctga tccccatgtc 4380

taccactggc agaccgtgat ggacgacacc gtgtccgcca gcgtagctca agccctggac 4440

gaactgatgc tgtgggccga agactgtccc gaggtgcgcc acctcgtcca tgccgacttc 4500

ggcagcaaca acgtcctgac cgacaacggc cgcatcaccg ccgtaatcga ctggtccgaa 4560

gctatgttcg gggacagtca gtacgaggtg gccaacatct tcttctggcg gccctggctg 4620

gcttgcatgg agcagcagac tcgctacttc gagcgccggc atcccgagct ggccggcagc 4680

cctcgtctgc gagcctacat gctgcgcatc ggcctggatc agctctacca gagcctcgtg 4740

gacggcaact tcgacgatgc tgcctgggct caaggccgct gcgatgccat cgtccgcagc 4800

ggggccggca ccgtcggtcg cacacaaatc gctcgccgga gcgcagccgt atggaccgac 4860

ggctgcgtcg aggtgctggc cgacagcggc aaccgccggc ccagtacacg accgcgcgct 4920

aaggaggtag gtcgagttta aactctagaa ccggtcatgg ccgcaataaa atatctttat 4980

tttcattaca tctgtgtgtt ggttttttgt gtgttcgaac tagatgctgt cgaccgatgc 5040

ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg 5100

ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 5160

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 5220

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 5280

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 5340

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 5400

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 5460

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 5520

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 5580

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 5640

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 5700

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 5760

aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 5820

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 5880

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 5940

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 6000

atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 6060

tcaatctaaa gtatatatga gtaaacttgg tctgacagcg gccgcaaatg ctaaaccact 6120

gcagtggtta ccagtgcttg atcagtgagg caccgatctc agcgatctgc ctatttcgtt 6180

cgtccatagt ggcctgactc cccgtcgtgt agatcactac gattcgtgag ggcttaccat 6240

caggccccag cgcagcaatg atgccgcgag agccgcgttc accggccccc gatttgtcag 6300

caatgaacca gccagcaggg agggccgagc gaagaagtgg tcctgctact ttgtccgcct 6360

ccatccagtc tatgagctgc tgtcgtgatg ctagagtaag aagttcgcca gtgagtagtt 6420

tccgaagagt tgtggccatt gctactggca tcgtggtatc acgctcgtcg ttcggtatgg 6480

cttcgttcaa ctctggttcc cagcggtcaa gccgggtcac atgatcaccc atattatgaa 6540

gaaatgcagt cagctcctta gggcctccga tcgttgtcag aagtaagttg gccgcggtgt 6600

tgtcgctcat ggtaatggca gcactacaca attctcttac cgtcatgcca tccgtaagat 6660

gcttttccgt gaccggcgag tactcaacca agtcgttttg tgagtagtgt atacggcgac 6720

caagctgctc ttgcccggcg tctatacggg acaacaccgc gccacatagc agtactttga 6780

aagtgctcat catcgggaat cgttcttcgg ggcggaaaga ctcaaggatc ttgccgctat 6840

tgagatccag ttcgatatag cccactcttg cacccagttg atcttcagca tcttttactt 6900

tcaccagcgt ttcggggtgt gcaaaaacag gcaagcaaaa tgccgcaaag aagggaatga 6960

gtgcgacacg aaaatgttgg atgctcatac tcgtcctttt tcaatattat tgaagcattt 7020

atcagggtta ctagtacgtc tctcaaggat aagtaagtaa tattaaggta cgggaggtat 7080

tggacaggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt 7140

gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca 7200

aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctct 7246

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

agttatggca gaactcagtg 20

<210> 17

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

ccccatccaa agtttttaaa gga 23

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

tgtggcagat gtcacagttt agg 23

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

caccgagtta tggcagaact cagtg 25

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 20

aaaccactga gttctgccat aactc 25

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 21

gaaggagcaa actgacatgg 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 22

tgcagtgggt ctttggggac 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 23

ttccaggaac ataagaaagt 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 24

gcagtctcag caaccactga 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 25

ggtcggagtg aacggatttg 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 26

ccatttgatg ttggcgggat 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 27

agatccgcca caacatcgag 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 28

gtccatgccg agagtgatcc 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 29

cctgctgtaa gtgccgtagt 20

<210> 30

<211> 18

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 30

ctaggggcac agcacgtc 18

<210> 31

<211> 26

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 31

aagttattag gtctgaagag gagttt 26

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 32

cccatcattc cgtcccagag 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 33

tgctgagttc tggcttcctg 20

<210> 34

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 34

tctaccaaga gagtgaccag cag 23

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 35

cacgccatcc tgcgtctgga 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 36

agcaccgtgt tggcgtagag 20

<210> 37

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 37

gtctccgctc gtcacttatc ct 22

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 38

ctagcagcct ttctttgtca gc 22

<210> 39

<211> 1266

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 39

atggatagag ttctgagcag agctgacaaa gaaaggctgc tagaacttct aaaacttccc 60

agacaactat ggggggattt tggaagaatg cagcaggcat ataagcagca gtcactgcta 120

ctgcacccag acaaaggtgg aagccatgcc ttaatgcagg aattgaacag tctctgggga 180

acatttaaaa ctgaagtata caatctgaga atgaatctag gaggaaccgg cttccaggta 240

agaaggctac atgcggatgg gtggaatcta agtaccaaag acacctttgg tgatagatac 300

taccagcggt tctgcagaat gcctcttacc tgcctagtaa atgttaaata cagctcatgt 360

agttgtatat tatgcctgct tagaaagcaa catagagagc tcaaagacaa atgtgatgcc 420

aggtgcctag tacttggaga atgtttttgt cttgaatgtt acatgcaatg gtttggaaca 480

ccaacccgag atgtgctgaa cctgtatgca gacttcattg caagcatgcc tatagactgg 540

ctggacctgg atgtgcacag cgtgtataat ccaaaacggc ggagcgagga actgaggaga 600

gcggccacag tccactacac gatgactact ggtcattcag ctatggaagc aagtacttca 660

caagggaatg gaatgatttc ttcagaaagt gggaccccag ctaccagtcg ccgcctaaga 720

ctgccgagtc ttctgagcaa cccgacctat tctgttatga ggagccactc ctatccccca 780

acccgagttc tccaacagat acacccgcac atactgctgg aagaagacga aatccttgtg 840

ttgctgagcc cgatgacagc atatccccgg acccccccag aactcctgta tccagaaagc 900

gaccaagacc agctggagcc actggaggag gaggaggagg agtacatgcc aatggaggat 960

ctgtatttgg acatcctacc gggggaacaa gtaccccagc tcatcccccc ccctatcatt 1020

cccagggcgg gtctgagtcc atgggagggt ctgattcttc gggatttgca gagggctcat 1080

ttcgatccga tcctagatgc gagtcagaga atgagagcta ctcacagagc tgctctcaga 1140

gctcattcaa tgcaacgcca cctaagaagg ctagggagga ccctgctcct agtgactttc 1200

ctagcagcct tactgggtat ttgtctcatg ctatttattc taataaaacg ttcccggcat 1260

ttctag 1266

<210> 40

<211> 1104

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 40

aataaatgca ctgttgggcc tatgctcaag atgggtagtg ttaattggtg gtggaactta 60

tctgatttca tgacttgctg gctacctaaa acaggtgagg agaaagccaa tgctatgtct 120

gggactggat gagcaagtac aacaaacaaa atgggcttaa agtatgagtg agagttatct 180

gaccgtaagg atgcaagtga gggggcctaa ggtttggaga ttaatattta atctcagatg 240

ctatactttg gtggtgtagc aaaagtctac aaatgggatg actgtaaaac tcagtagatc 300

cgtgcttttt aacctatctc ccttcatcag gaaattgcga cacaaagatc tttagtaata 360

acacgcagtc tcaatgcata aaatcaggct taggtgttgc ctggactcat ttcccatctc 420

caccccacta taattatttt gtgacacaaa ctcaagactg tgggaatata gagaaattgg 480

gctcgtcctc gtacacctgc tcaatcccct gcaggacaac gcccaagaat caggttaagc 540

cagggcaaaa gaatcccgcc cataatcgag aaggagcaaa ctgacatgga ggcgatgacg 600

agatcgcggg ggagggaggg atttttctag gcccagggcg gtccttagga aaaggaggca 660

gcagagaact cccataaagg tattgcggca ctcccctccc cctgcggaga agggtgcggc 720

cttctctccg cctcctccac tgcagctccc tcaggattgc agctcgcgcg ggtttttgga 780

gaacatgcgc ctcccaccca caagccagca ggaccgaccc cccactcctt cctccacccc 840

ccacccccac gggtccgaga gcaggtagag ggctagtctc gtccttcagg cggcggacgc 900

ccagggcgga gccgcagtca ccaccaccca gaagcctcgg cccggcagcc cgcccccgcc 960

tcctgcgcgc gcttcctgcc acgttgcgca ggggcgaggg gccagacact gcggcgctgg 1020

cctcggggag ggccgtacca aagaccgcct ccctgccgac tcgcgtagtg gtttcgctca 1080

tttgggaccc aagccaataa caag 1104

<210> 41

<211> 1056

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 41

tgctctctct cctgccccct tcacctgcgt gccctcctca ttctccctct gtgccacctc 60

tggccttgca ctgtaggctc tctcttgggg atgtttctcc ttctccacac acttctcttt 120

cactctgtcc tcttgctttg tgtgggcctg cagcgttacc cttttttctg ggcacactca 180

gagcaccctc ctctttctgg ttctgggcca cctgtctgtc ctcgggtcat cttgctctct 240

ctgcctggat gccctcctgt ggctttgggc agcttctccc tccttcagag tgcaccgcca 300

gttctcctag gcccggtcac ttccccttcc caggggacct agagccctgc taggtcctct 360

ctctccacaa cctgggcccc caaacctttc caaaacacct tgctttctgc ctccattggt 420

cttgtgttcc agagccagag tcactatatg tcccagaacc aggattccct ctggttctga 480

gggcttttat cgcatcccct gcctggctgc agtgggtctt tggggacagg ccacagaaga 540

gcctctactc ctccctctgt ccccgaggct gtctccctcc cagtcttccc agctcaggcc 600

agtccccagg cctctcttcc ctgccagagc ccgtcaggtt cggttacttt ggggcccaga 660

gaggaccctg tgaaggaagc gtgggtaggg gcacgggaat ggggaggatg cctgaagagg 720

cccccttagc cagaagagga gcagaagagg agcaggtacc cagaagagga gcagttcagg 780

gaaatagaag agtcccgagc tctttttttt tttttttttt atttcttttc ttttcttttc 840

tttttatggc agcatccgtg gtatatggag gttcccagcc taggggtcag atcatacctg 900

caactgccag cctacaccac agccacagca ctcaggatcc gagctgcatc tgcggcttac 960

gccacaggtc acagcaacgc tggatcctta acccactgaa tgaggccagg gattgaacct 1020

gcaacctcat gcacactatg ctggggtctt aatcgg 1056

<210> 42

<211> 1108

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 42

acttcctcct gcccttaccc tttatctggc tcttagctcc taaaaactgc attattagct 60

tcctcttttg cctctactct tactcaacca aaattgtttt aagatctgtg gatctagctt 120

ctgctgtgct attcttagga acacttttat ttcctcttag ctccatctca ccagttattg 180

gctaatggct ttgcttggta cctacatctg tacatttctt tcgtactagc ttctagactg 240

aaaaaggact gttggttcaa catgaaaggg aaggaggtaa aagaggacac acaggaaaga 300

tggattggga ttcaggtctc tgctgttgtt acttgagatt gctttctaga ttctacttgt 360

ggaaacaaaa agcctttgcg agaattctaa actggagtat ttctgtaatt gaggagtctt 420

gctcagcaaa tcccacttag gggactaatg aagtaccagg aagagacaga ccatgctcaa 480

tccacaaagc caggttttac tgaaatgtga cctactttct tatgttcctg gaagtttaga 540

tcagggtggg cagctctggg ttttataggc tacactgtta acactcaggc tgttttctac 600

cgtttagtca aaatatagtc accttgcctg cttcacctgt ccatcagaga atggcctcat 660

taattgactc tctagtatga agtcaaagta gctttggtgg ccctaaatgg acaagtatca 720

agagactggg tgaattgagg agcttgagac tgtcacctca gatcgaaaag actgaaaaat 780

cacctcagat caaaaagact gaaaaatctt cagtctggaa aggggactca aaaccataat 840

tagagtattc tggtagaatc cttttctcca ctgttattca tacagttaag gtgaataact 900

aaaagtaatt gtgagctgag gagtaagata caacacacaa ggaatcagtt aacagagtct 960

cgagtgaaat tataaatgga aagaattatg acttgaatca taactctgag gccccatttt 1020

ccctaacaac ttttgtccca ataaacgtgg gtatttgttt gggagaaact atcatataca 1080

tgattaccca gtaaacagac tgtttact 1108

<210> 43

<211> 1089

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 43

actttgtacc tattttgtat gtgtataata atttgagatg tttttaatta ttttgattgc 60

tggaataaag catgtggaaa tgacccaaac caatcttgca ctggcctcct gatttccttc 120

cttggagacg gagggagggg gagacctggg ggagggcgct tggggggggg tgggctctct 180

tctttctgcg ctcccccccc ccacctccaa caccttgacg acccctcctg cttccgcttg 240

cctttctcag gctttaacac tttctcctcg ccctctcagc atgcgcatgc gcgtgcctct 300

acctcccccg cacatcctgg cctgcccacc ctgaatgtcc tggcccagcg atgccaccaa 360

ctctctcgct ccgtccacgg ctggggaggg gggcactctg cagggttggg gggcactggg 420

aggctgggtt gggtgaggga ggggtgcctg ggcccccacc ccccagcaag ttctctccct 480

aggcgaactg gagggtcgtc tggcctcttg agccttgttg ctggctctga gctctaccaa 540

gagagtgacc agcaggaccg caccatcagt ggttgctgag actgcgtggg ggcccaagga 600

gacctggaga aaggaatgct tcctgctcct tcttctgggg ccccaggaga gccttcccag 660

ggccttggag aggtgctgtc cagggactaa ccctgtgctc taggaaggct gcaggccctg 720

accagctggg caggtcctgg gtccctcctg gccttctaag ttccccaaac atgagacctc 780

tgggtgtggg gtggcctggg gaggtcattt tgcccaggcc ctacctcctg cccattccta 840

acccttttta aaaatctgtg cgtcctcttc ttccttcttc tccctccctt cccttttcgc 900

tcaccctctg ctgctggcct gagagccgga ggcccccagg gggaaggcga ctggtctcct 960

ccccagtctc agggaaggga gacagagaat ccaggaagcc agaactcagc agacgaagca 1020

cccagggacc tagagatggg ttgaaaagtt gacagctgtc ccacctgcct cccaaggtct 1080

cagggccta 1089

Claims

1. A construction method of a pig cell for expressing PyMT is characterized in that a nucleotide sequence for encoding the PyMT is inserted into a pig safe harbor site to obtain a polypeptide expressing SEQ ID NO:14, wherein the nucleotide sequence of the coded PyMT is shown in SEQ ID NO:39, wherein the nucleotide sequence encoding the PyMT is regulated and controlled in the pig cells by an exogenous promoter, the exogenous promoter is MMTV-LTR, and the pig safe harbor site is selected from the pig ROSA26, AAVS1, H11 or COL1A1 safe harbor site;

The construction method comprises the steps of co-transfecting a safe harbor site vector, an sgRNA vector and a Cas vector into pig cells, wherein the Cas vector comprises a nucleotide sequence for encoding Cas9 protein, EGFP and Puro resistance protein;

Inserting a nucleotide sequence encoding a PyMT into a pig safe harbor site using a safe harbor site vector, the safe harbor site vector comprising a nucleotide sequence encoding a PyMT and a safe harbor site vector backbone comprising a 5 'homology arm and a 3' homology arm of the safe harbor insertion site, the nucleotide sequence encoding a PyMT being located between the 5 'homology arm and the 3' homology arm, the safe harbor site vector backbone being selected from any one of the following:

a) The ROSA26 safe harbor site vector skeleton, the 5' homology arm of which is shown in SEQ ID NO:5, the 3' homology arm is shown in SEQ ID NO:6 is shown in the figure;

b) AAVS1 safety harbor site carrier skeleton, its 5' homology arm is as SEQ ID NO:7, the 3' homology arm is shown as SEQ ID NO: shown as 8;

c) H11 safe harbor site carrier skeleton, the 5' homology arm of which is shown in SEQ ID NO:9, the 3' homology arm is shown as SEQ ID NO:10 is shown in the figure;

Or D) a COL1A1 safe harbor site carrier skeleton, wherein the 5' -homology arm is shown in SEQ ID NO:11, the 3' homology arm is shown as SEQ ID NO: shown at 12;

The sgRNA vector comprises a sgRNA targeting ROSA26, AAVS1, H11 or COL1A1 safe harbor site, wherein:

The nucleotide sequence of the sgRNA targeting the ROSA26 is shown in SEQ ID NO: as indicated by the reference numeral 21,

The nucleotide sequence of the sgRNA targeting AAVS1 is shown in SEQ ID NO: as indicated by the reference numeral 22,

The nucleotide sequence of the sgRNA targeting H11 is shown in SEQ ID NO: as indicated by the numeral 23,

The nucleotide sequence of the sgRNA targeting COL1A1 is shown in SEQ ID NO: shown at 24.

2. The construction method according to claim 1, wherein the nucleotide sequence of 500bp each in the region of the ROSA26 safety harbor site and upstream and downstream thereof is as set forth in SEQ ID NO:40, and the nucleotide sequence of 500bp respectively at the AAVS1 safe harbor site region and the upstream and downstream thereof is shown as SEQ ID NO:41, and the nucleotide sequence of 500bp respectively at the upper and lower reaches of the H11 safe harbor site region is shown as SEQ ID NO:42, the nucleotide sequence of 500bp of each of the COL1A1 safe harbor site region and the upstream and downstream thereof is shown as SEQ ID NO: 43.

3. The method according to claim 1, wherein the nucleotide sequence of MMTV-LTR is as set forth in SEQ ID NO: 15.

4. The method of claim 1, wherein the porcine cells are porcine fibroblasts or breast cells.

5. The method of construction of claim 1, wherein the Cas vector further comprises an EF1a promoter, a WPRE element, and a 3' ltr sequence element.

6. The construction method according to claim 1, wherein the Cas vector has the nucleotide sequence of, in order from 5 '-3': CMV enhancer, EF1a promoter, nuclear localization signal, nucleotide sequence encoding Cas protein, nuclear localization signal, nucleotide sequence encoding self-cleaving polypeptide P2A, nucleotide sequence encoding EGFP, nucleotide sequence encoding self-cleaving polypeptide T2A, nucleotide sequence encoding Puro resistance protein, WPRE sequence element, 3' ltr sequence element and polyA signal sequence element.

7. The method of claim 1, wherein the Cas vector has a nucleotide sequence set forth in SEQ ID NO: 2.

8. A method for constructing a breast cancer model pig, comprising transferring pig cells obtained by the construction method according to any one of claims 1 to 7 into enucleated pig oocytes to obtain a breast cancer model pig.

9. Use of a pig cell obtained by the construction method of any one of claims 1 to 7 in the preparation of an animal model of breast cancer, or in the screening of a medicament for the treatment of breast cancer, or in the study of the pathogenesis of breast cancer.

10. Use of a breast cancer model pig obtained by the construction method of claim 8 in screening for a drug for treating breast cancer or in studying the pathogenesis of breast cancer.