CN112626119A - Construction method of human CYP2D6 x 10 transgenic mouse model - Google Patents

Construction method of human CYP2D6 x 10 transgenic mouse model Download PDF

Info

Publication number
CN112626119A
CN112626119A CN202011499244.0A CN202011499244A CN112626119A CN 112626119 A CN112626119 A CN 112626119A CN 202011499244 A CN202011499244 A CN 202011499244A CN 112626119 A CN112626119 A CN 112626119A
Authority
CN
China
Prior art keywords
positive
mouse
hyp
gene
mice
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202011499244.0A
Other languages
Chinese (zh)
Inventor
陈冰冰
钱建畅
胡国新
李军伟
肖健
林丽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wenzhou Medical University
Original Assignee
Wenzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wenzhou Medical University filed Critical Wenzhou Medical University
Priority to CN202011499244.0A priority Critical patent/CN112626119A/en
Publication of CN112626119A publication Critical patent/CN112626119A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Environmental Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a construction method of a human CYP2D6 x 10 transgenic mouse. According to the invention, by utilizing a homologous recombination principle and adopting an ES targeting mode, an expression frame of hYP 2D6 x 10 is knocked in at a fixed point of an exon4-5 site of a Cyp2D22 gene, and a Cyp2D gene cluster is knocked out by a Crispr/Cas9 mode. A targeting vector is prepared, and the vector is linearized and then used for electrotransfection of ES cells. The positive clone of correct homologous recombination is obtained through long-fragment PCR identification. Positive ES cell clones were expanded and injected into blastocysts of C57BL/6J mice to obtain chimeric mice. Compared with non-transgenic mice, the human CYP2D6 x 10 transgenic mice bred by the invention can express human CYP2D6 x 10 type genes (Chinese population dominance ratio alleles), and can be used for carrying out relevant researches on pharmacokinetics and pharmacodynamics of substrate drugs instead of human.

Description

Construction method of human CYP2D6 x 10 transgenic mouse model
Technical Field
The invention belongs to the technical field of biology, and relates to a construction method of a CYP2D6 x 10 transgenic mouse model, in particular to a method for expressing main genotype CYP2D6 x 10 in a CYP2D6 family of Chinese population in a transgenic mouse.
Background
CYP2D6 is one of the most prominent subtypes of genetic polymorphisms in the CYP450 family, mediating at least 20% of the in vivo elimination of clinical drugs (Gaedigk A, Sangkuhl K, Whil-Carrillo M, Klein T, Leeder JS.prediction of CYP2D6 phenotype from genetic species across world positions. Gene. 2017; 19(1): 69-76; Zhou SF, Liu JP, Lai XS. Substrate specificity, inhibition and regulation of human cytochromes P4502D6 and microorganisms in drug discovery Medium chem. 2009; 16(21): 26805). At present, 141 CYP2D6 alleles (containing no non-amino acid coding region variation) have been reported to encode proteins with a wide range of enzymatic activity alterations, manifested as loss, reduction, normality or increase in activity. The metabolic phenotype varies significantly among individuals and populations, with CYP2D 6-1 not being the majority in east Asia, but CYP2D 6-10 alleles having up to-50% frequency as seen by the frequency of occurrence of the alleles (Qian JC, Xu XM, Hu GX, Dai DP, Xu RA, Hu LM, et al. genetic variations of human CYP2D6 in the Chinese Han publication. Pharmacogenomics.2013; 14: 1731-43; Dai DP, Geng PW, Wang SH, Cai J, Hu LM, Nie JJ, et al. in vitro functional of 22 neural differential CYP2D6 allelic in the genomic DNA library III, Tocol III, To III 11-43). CYP2D6 x 10 is the main reason of weak metabolism of Chinese population, and the research on the substrate pharmacokinetic change is an important way to grasp the curative effect difference among drug individuals.
At present, CYP450 enzyme activity research is mainly in vitro and lacks in vivo research models, so that related data influencing different metabolic phenotypes are often lacked in the preclinical research stage of medicaments, nearly 40% of new medicaments fail to be researched and developed (the medicament generation parameters do not meet the declaration requirements), and nearly 5% of people are hospitalized due to adverse drug reactions (the blood concentration is too high or the medicaments interact with each other); even a large proportion of the population does not respond to drugs (Novalbos J, Lopez-Rodriguez R, Roman M, Gallego-Sandin S, Ochaa D, Abad-Santos F. effects of CYP2D6 genes on the pharmaceuticals, pharmaceuticals J. experiments of risperidone in health volnters. J. clinin pharmaceuticals.2010; 30(5) 504-11; Kirchheiner J, Keulen JT, Bauer S, Roots I, Bromomoner J. effects of CYP2D6 gene amplification on the pharmaceuticals and magnetics of clinical chemistry J. experiments J. reactions of clinical chemistry J. 2008. 9. and 10. faecal J. experiments of the pharmaceuticals J. 9. experiments of clinical application J. 9. experiments, Sri. 10K. J. experiments and magnetics of clinical chemistry J. 9. faecal. J. experiments, European bacteria J. 9. faecal. J. experiments, and K. 9. experiments of fungal strain J. 9. K. F. 9. experiments, and K. 9. experiments of fungi. K. A. of the present in the family of the family members, such as "family of the family. The mouse used for in vivo research has certain difference between the type of in vivo metabolic enzyme and a human body, so that a majority of CYP2D6 x 10 humanized transgenic mouse models in Chinese population are constructed, the correlation research of metabolic phenotype and clinical Drug effect is carried out through the new relation of CYP2D6 gene-functional phenotype, a theoretical basis is provided for finding potential Drug-Drug Interaction (DDI), a data support is provided for improving the curative effect (dose dependence) of a substrate Drug, and the mouse has wide application prospect and value.
Disclosure of Invention
The invention aims to provide a method for constructing a human CYP2D6 x 10 transgenic mouse model, which aims to solve the problem that the type of metabolic enzymes in the body of the existing mouse for in vivo research is different from that of the human body.
The method for constructing the human CYP2D6 x 10 transgenic mouse provided by the invention can comprise the following steps: the method comprises the following steps: knocking in an expression frame of human CYP2D6 x 10 (hYP 2D6 x 10) at a fixed point between mouse Cyp2D22 gene exon4-5 by utilizing a homologous recombination principle, obtaining a positive clone, knocking out a Cyp2D gene cluster, and obtaining an hYP 2D6 x 10 gene targeting vector; obtaining Cyp2dKO positive ES cells after targeting, injecting the cells into blastocysts of a mouse through amplification, and then transferring the blastocysts into a surrogate mouse for breeding to obtain a chimeric mouse;
the nucleotide sequence of hYP 2D6 x 10 is sequence 1 in the list;
the sequence of the hYP 2D 6-10 gene targeting vector is shown as sequence 2 in the list.
Specifically, the vector 1 applied in ES targeting comprises a 3.0kb 5 'homologous arm, hYP 2D6 promoter-hYP 2D6 10genomic DNA-polyA, PGK-Neo-polyA, a 3.0kb 3' homologous arm and an MC1-TK-polyA negative selection marker, and the vector 2 comprises U6 promoter-sgRNA 1; vector 3 contained U6 promoter-sgRNA 2. Wherein the nucleotide sequence of the vector 1 is shown as sequence 2 in the list and consists of 27205 nucleotides. After restriction by EcoRI, 5 characteristic bands were present: 10478bp, 8673bp, 5617bp, 2177bp and 260 bp.
The ES targeting positive cells are prepared according to the following steps: after linearization of the vector with restriction enzymes, C57 BL/6X 129S3 ES cells were transfected by electrotransfer. Screening by using antibiotics and acquiring positive clones by combining a PCR technology. More specifically, the 5 'homology arm and the 3' homology arm are amplified separately using PCR technology. Among them, the 5' arm homologous positive clone should amplify a 3.6kb fragment, while the negative clone should have no product. The positive clone of 3' homology arm should be amplified to obtain 6.0kb fragment, and the negative clone should have no product.
In the invention, after the CYP2D6 x 10 positive ES cells are obtained, the CYP2D gene cluster of a mouse is specifically knocked out by utilizing the Crispr/Cas9 technology, and KO positive clone ES cells are obtained by screening.
More specifically, KO-positive ES cells were screened by PCR, and positive clones having a 2.2kb fragment were obtained, whereas negative clones had no product.
In the present invention, the strain of the mouse is C57 BL/6J. The ES cells obtained by the above preparation were injected into blastocysts of mice by microinjection, and chimeric mice of F0 generation were obtained after birth. If it is desired to obtain homozygous CYP2D6 x 10 mice, chimeric mice are crossed.
Compared with the prior art, the invention has the beneficial effects that:
the invention can overcome the problem that the type of metabolic enzyme in the body of the existing mouse has certain difference with the human body by constructing the human-derived CYP2D6 x 10 transgenic mouse model, and the obtained human-derived CYP2D6 x 10 transgenic mouse can express human CYP2D6 x 10 type gene and can be used for carrying out relevant researches on pharmacokinetics and pharmacodynamics of the substrate drug of people with the CYP2D6 x 10 genotype prevailing in China.
Drawings
Figure 1 is a schematic of the strategy for constructing humanized CYP2D6 x 10 transgenic mice.
FIG. 2 shows the electrophoresis of homologous recombination vector plasmid and enzyme digestion identification.
FIG. 3 is a schematic representation of the ES cell identification strategy.
FIG. 4 shows the electrophoresis result of PCR identification of homologous recombination positive ES cells.
Detailed Description
The invention is further illustrated in the following examples. These examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Experimental mice C57BL/6J and ICR were purchased from Shanghai, Inc. of Modular Biotechnology, Inc. Various restriction enzymes, T4 DNA ligase, T4 DNA polymerase, Taq enzyme and PCR-related reagents were purchased from TaKaRa or NEB. Agarose gel recovery kits were purchased from Axygen bio-inc. Cloning of recombinant reagents In-Fusion HD Cloning kit was purchased from Takara. Cell culture related reagents: DMEM medium (high-sugar, ES cell grade), fetal bovine serum (ES cell grade), G418, Gancycovir, LIF, streptomycin, trypsin, etc., required for ES cell culture were purchased from Gibco BRL, Sigma, and Chemicon, respectively.
Example 1 the present invention relates to a method belonging to the knock-in of the humanized CYP2D6 x 10 gene in situ
As shown in FIG. 1, the present invention first constructed ES cell targeting plasmids comprising 3.0kb 5 'homology arm, hYP 2D6 promoter-hYP 2D6 genomic DNA-polyA, PGK-Neo-polyA, 3.0kb 3' homology arm and MC1-TK-polyA negative selection marker. And (3) carrying out ES cell targeting after linearization of the targeting plasmid, carrying out homologous recombination of the targeting vector and a C57BL/6J mouse ES cell genome to obtain a gene recombinant ES cell with a human CYP2D6 gene inserted between the No. 4 exon and the No. 5 exon, and screening by G418 pressure to obtain a resistant ES cell. Positive ES cell clones were screened for double-arm correct homologous recombination by PCR and sequencing. After obtaining positive clone, the Cas9-gRNA vector aiming at the target gene is transfected into the positive ES cell clone of correct homologous recombination, namely, the Cas9 technology is utilized to knock out the mouse cyp2d gene cluster so as to eliminate the influence of endogenous homologous protease of the mouse on the human protein. Cloning the knocked-out positive ES cells, and performing blastocyst injection to obtain the chimeric male mouse. Chimeric male mice were mated with Flp female mice, and offspring were genotyped to obtain F1-generation mice that were positive for removal of the Neo gene.
Example 2 validation of targeting plasmids by restriction enzymes or Gene sequencing
As shown in FIG. 2, targeting plasmids were constructed and the target sequences were verified by restriction enzyme and gene sequencing, respectively, to ensure the correctness of the sequences. The enzyme digestion reaction system was configured as shown in the following table:
Reaction Component Volume(μl)
ddH2O 15.9
10×Buffer 2
EcoRI 1.5
Plasmid DNA 0.6
Total 20
and (3) uniformly mixing the system, putting the system into a water bath kettle at 37 ℃, reacting for 2 hours, adding a proper amount of Gel loading buffer, uniformly mixing, performing agarose Gel electrophoresis, and photographing after strip separation to obtain 5 strips with 10478bp, 8673bp, 5617bp, 2177bp and 260bp (small segments can not be seen due to the fact that the strips are low in content). The plasmid with correct enzyme digestion verification is sent to a sequencing company for sequencing.
Example 3 screening of the resulting ES cell clones by PCR
As shown in fig. 3, PCR identification protocol for homologous recombination positive clones: after extracting the genomic DNA of the resistant ES cell clone, homologous recombination positive clones were screened by long-fragment PCR, and the primer positions are shown in FIG. 3. 3.6kb fragment should be amplified by 5' arm homologous recombination positive clone, and no product should be generated by negative clone; the 3' arm homologous recombination positive clone should amplify 6.0kb fragment, and the negative clone should have no product. KO positive clones should amplify a 2.2kb fragment, and negative clones should be product-free.
Example 4 cloning identification of homologous recombination-Positive ES cells
(1) The PCR reaction system and conditions for the 5' homology arm of ES cells were as follows:
primer information:
Primer Sequence 5'-->3' Primer Type
I GAATGGGGACCGCTTGTTGTGC Forward
II GGTGGCTCGGTCCTGGGTTTTC Reverse
reaction system:
Reaction Component Volume(μl)
ddH2O 13.2
GXL PCR Buffer 2
2.5mM dNTP 2
Primer I/II(10pmol/μl) 0.5
GXL DNA Polymerase* 0.8
genomic DNA 1
Total 20
PrimeStar GXL (TaKaRa, Code No: R050A) reaction conditions:
Step# Temp(℃) Time Note
1 98 2min -
2 98 10sec -
3 61 15sec -
4 68 3min repeat steps 2-4for 34cycles
5 68 10min -
6 12 - hold
(2) the PCR reaction system and conditions for the 5' homology arm of ES cells were as follows:
primer information:
Primer Sequence 5'-->3' Primer Type
III CTGCCCAGGGAACGACACTC Forward
IV GGGCCAACGACATTCCTATCTTT Reverse
reaction system:
Reaction Component Volume(μl)
ddH2O 13.2
GXL PCR Buffer 2
2.5mM dNTP 2
Primer III/VI(10pmol/μl) 0.5
GXL DNA Polymerase* 0.8
genomic DNA 1
Total 20
*PrimeStar GXL(TaKaRa，Code No：R050A)
reaction conditions are as follows:
Step# Temp(℃) Time Note
1 98 2min -
2 98 10sec -
3 61 15sec -
4 68 3min repeat steps 2-4for 34cycles
5 68 10min -
6 12 - hold
(3) KO identification method
Primer information:
Primer Sequence 5'-->3' Primer Type
V AAACCATGGGATCGGCCATT Forward
VI CTACAGGCCTCTGGGACTCT Reverse
reaction system:
Reaction Component Volume(μl)
ddH2O 13.2
GXL PCR Buffer 2
2.5mM dNTP 2
Primer V/VI(10pmol/μl) 0.5
GXL DNA Polymerase* 0.8
genomic DNA 1
Total 20
*PrimeStar GXL(TaKaRa，Code No：R050A)
reaction conditions are as follows:
Step# Temp(℃) Time Note
1 98 2min -
2 98 10sec -
3 61 15sec -
4 68 3min repeat steps 2-4for 34cycles
5 68 10min -
6 12 - hold
after the reaction, a certain amount of PCR product was taken and a band of a specific size was detected using 0.8% agarose gel.
Sequence listing
<110> Wenzhou university of medical science
<120> method for constructing human CYP2D6 x 10 transgenic mouse model
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1494
<212> DNA
<213> human cytochrome P4502D6 x 10 (Homo sapiens)
<400> 1
atggggctag aagcactggt gcccctggcc gtgatagtgg ccatcttcct gctcctggtg 60
gacctgatgc accggcgcca acgctgggct gcacgctact caccaggccc cctgccactg 120
cccgggctgg gcaacctgct gcatgtggac ttccagaaca caccatactg cttcgaccag 180
ttgcggcgcc gcttcgggga cgtgttcagc ctgcagctgg cctggacgcc ggtggtcgtg 240
ctcaatgggc tggcggccgt gcgcgaggcg ctggtgaccc acggcgagga caccgccgac 300
cgcccgcctg tgcccatcac ccagatcctg ggtttcgggc cgcgttccca aggggtgttc 360
ctggcgcgct atgggcccgc gtggcgcgag cagaggcgct tctccgtgtc caccttgcgc 420
aacttgggcc tgggcaagaa gtcgctggag cagtgggtga ccgaggaggc cgcctgcctt 480
tgtgccgcct tcgccaacca ctccggacgc ccctttcgcc ccaacggtct cttggacaaa 540
gccgtgagca acgtgatcgc ctccctcacc tgcgggcgcc gcttcgagta cgacgaccct 600
cgcttcctca ggctgctgga cctagctcag gagggactga aggaggagtc gggctttctg 660
cgcgaggtgc tgaatgctgt ccccgtcctc ctgcatatcc cagcgctggc tggcaaggtc 720
ctacgcttcc aaaaggcttt cctgacccag ctggatgagc tgctaactga gcacaggatg 780
acctgggacc cagcccagcc cccccgagac ctgactgagg ccttcctggc agagatggag 840
aaggccaagg ggaaccctga gagcagcttc aatgatgaga acctgcgcat agtggtggct 900
gacctgttct ctgccgggat ggtgaccacc tcgaccacgc tggcctgggg cctcctgctc 960
atgatcctac atccggatgt gcagcgccgt gtccaacagg agatcgacga cgtgataggg 1020
caggtgcggc gaccagagat gggtgaccag gctcacatgc cctacaccac tgccgtgatt 1080
catgaggtgc agcgctttgg ggacatcgtc cccctgggtg tgacccatat gacatcccgt 1140
gacatcgaag tacagggctt ccgcatccct aagggaacga cactcatcac caacctgtca 1200
tcggtgctga aggatgaggc cgtctgggag aagcccttcc gcttccaccc cgaacacttc 1260
ctggatgccc agggccactt tgtgaagccg gaggccttcc tgcctttctc agcaggccgc 1320
cgtgcatgcc tcggggagcc cctggcccgc atggagctct tcctcttctt cacctccctg 1380
ctgcagcact tcagcttctc ggtgcccact ggacagcccc ggcccagcca ccatggtgtc 1440
tttgctttcc tggtgacccc atccccctat gagctttgtg ctgtgccccg ctag 1494
<210> 2
<211> 27205
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gaattctcat gtttgacagc ttatcatcga taagctgcgg ccgcaaaggc cgcggtcgac 60
aagctaacca ttagagatgt gaggcattca ccagagaaga ctactcagat atagatttaa 120
gccaatagaa gctttattag ctggctggtg acaacactgg gtgctcagaa tcccagtata 180
gtcctgagct gtactcaggg tgagctttta ccaacaacta tatcatgtat tgacacactt 240
cagttagcaa gaacagttag ctagaagaac tatcgaagtc aaaaagcaag attactactt 300
ttagggactt tcccagaact atggaatttg atggattagg tctttgtttt cattatgaca 360
ggaactgctg tctatgtgca gagttttatg gtacttccat catggactca gctgtgctaa 420
ggtctgagga ctttctaagg tctgggagcc tgtttcaaca tcaagcttgc aggtcctacc 480
aaccctggtt acagaggcac tttcttactg ggcatgggcc tggacaacag tatccctgtt 540
gggtcatgga gttagtgttg acaaagaagc ttaggaggta aacccctgct tctggactcc 600
cttaaagcct tttagagaag catgtggtac ccaacaggaa accttaggca cttaacctgc 660
atatcatttc cagcttcccg gacttctcta catctttggg gatgccatat aagacgttgt 720
gatgatgctg gctaagcagc tagtcttggt tgtgtggtag gtctgaaatg ggggcatcta 780
ggagagagag gtcctgccct tccttcatct atgtaactta cactgtcaca tcttagcagt 840
cccttctggc catccttcca gacgttggca tctatgtata aacattatca atttgaggga 900
aaaagtgggg gaagggtaaa ggcaagaatg gtatcatttc tgacagggca gtccttctac 960
tggggtcaaa tagtcaagtc agaataagca gggaaagtgg atgctcatgg gaaggctgcc 1020
taggtccctt acagccagga gatctcatag ggatgattta gtttagagta aaatgcttta 1080
ttgcacatta ggaccccagc aagcacagtg agtgagatgt catacatcct cttagcgggg 1140
caaagcacag agctggtagg ggcatggggt tgtcagagcc ctaaagacgc catggtcgct 1200
gggctggggc tgtccatcgg gcactgagat gctaaagcgc tgcaggaggc aggtgaagaa 1260
gaggaagagc tccatgcggg ccaggggctc ccccaggcat gatctgcggc ctgtgggtag 1320
gagagggttc agtgagccag gggcctggct tcacaattca aagcattctg ggaagaggaa 1380
gggaagccag gcatcccccc aggcacctgc tgagaatggc atgaaggcct caggcttcac 1440
aaagtggccc tgggcatcca ggaagtgttc aggatggaag cagaggggct tctcccagac 1500
agtctcatct tttagcgcgg aggacaggtt ggtgatgagg gtcgtcccct ggcagaaggc 1560
acatggtgtg aggttggact ggggtggggc cgtctagact ctgatcacca agctcacatg 1620
cgtggtctct acctcacaga ctggatcctt tcatataacc ctagccctag tggcttctta 1680
gctgtcttta atactatgac agggagaagt gacaagtgac agatgtccac actgtcacct 1740
gtgtctgtag gtagtggctg ttacaaccac tcctgccata ttttaccttg cttgccttga 1800
agattctagc gtacagttcc tgctactgtt tctcttccca tacttaccaa gtagaagttg 1860
gttctgtgga attgagaatt gagttgccta agttccagtc ctgtcttaag tggacagctc 1920
aggccactag ctagctggca acaccgacta gacattggag ggtcccaggc ctaccttagg 1980
gataaggaag ccctgtagtt caatgtcacg agaagtcttg tgaggtacac caagagggag 2040
aatgtctgca aagcgctgca cctcatgaat gacagcattg gtgtagggca tgcgagcctg 2100
gtctgccatc tctggacact gcacctgccc tatgacttca tcgatttcct gttgtactcg 2160
gcctggaagg acagcatcac ctcagttaag gagcctcagg gggtggtgcc ccttgtccct 2220
ccactacctc tatggtaagg gctgggttca tgggagaatg atagccattg tccttgctgg 2280
agtgctggga gccacaaatg tcctaagtaa gtttaaggac tgtgcctcta ccctcctcct 2340
cctctcatcc cagtcccggc caggctcacg ctgcacatct ggatgcagga tcatgagcat 2400
cagggcccag gacagtgtgg ttgaggtggt caccatccct gcagagaaca ggtcacccac 2460
taccgtgcgc aggttctcat cattgaagct gctctcagga ttccccttgg cctgggccag 2520
gcagaaagaa tgggctcagt aggaagagga agtcccacat ggcttcccat tgtccccagg 2580
gcagcccaag gtgttgagtc acttggtgat acacaggtct ctatccatct ggcctcacag 2640
ctcacaggac ctcagcccac agcccctggc cacatctctt ggagctgcca atcaccttct 2700
ccacctcagc caggaaggca tcagtcaaat ctcggggtgg ctgggtaggg tcccaggtcg 2760
tcttgtgttc agccagcagc tcatccaaca tggtaacaaa ggcccttttc ccagggaaga 2820
ccttgccaac cagccccggg atgcgcagga gcatcgggaa cacattcagg aactgtgtga 2880
gcagtagaga aggaccaata gagccctggc tgcctcctgg ctgtgctctc ttggaacagc 2940
ccaggtccca gcctcctcca ggttagatta gtgacacagt ctctccctgc cagcctttag 3000
ccttcccatt tgccctgagc ttccagaagg gctgtcatat tgtggaaaca gatgtgtgtg 3060
gctgcttaat taaggtcagc tccttcctct cgaattttca gggagctata tgccagggct 3120
acctcccagg gctcagtcgg caggtgccag aacattccct gggaaggccc caggaaaacc 3180
caggaccgag ccaccgccct cagcctgtca ccttgtgtcc aaaattggtg ggttcttggt 3240
ctcactgact tcaagaatga agccgtggac cctcacggtg agtgttacag ttcttaaaga 3300
tggtgtgttc agagtttgtt ccttctgatg ttaagacgtg ttcagagttt cttccttctg 3360
gtgggtgcgt ggtcttgctg gcttcaggag tgaagctgca gaccttcaca gtgagtgtta 3420
cggctcttaa ggctgcacgt acggagttgt tcattcttcc tggtgggttt gtggtctcac 3480
tggcctcagg agtgaaactg cagtccttcc agtgttacaa ctcataaagg cagtgtggac 3540
ccaatgaggg agcagcagca gcaagactta ctgcaaacag caaaagaatg atggcaacca 3600
ggttgccgct gctacttcag gcagcctgct tttattccct tatctgaccc ccacccacat 3660
cctgctgatt ggcccatttt acagacagtg gattggtcca cttacagaga gctgattggt 3720
gcatttacaa tccctgagct agacacagag tactgattgg tatatttaca aaccttgagc 3780
tagacacaga gtgctgaatg gtgtatttac aatcccttag ctagacataa aggttgtccc 3840
agtccccact agattagcta gatagagtag acagagagca ctgattggtg cgtttacaaa 3900
ccttgagtta gacacagggt gctgactggt gtgtttacaa accttgagct agacacagag 3960
tgctgattgg tgtatttaca atcttttagc tagaaataaa ggttccccaa gtccccacca 4020
gattagctag atagagtgct aattggtgca tgcacgaacc cggagctaga cacagagtgc 4080
tgattggtgc atatacaatc ctctggctag acataaaagt tctccaagtc cccacctgac 4140
tcaggagccc agccagcttc gcctagtgga tcctatgcca gggccacagg cagagctgcc 4200
tgctagtccc acaccgggca cctgtactcc tcagcccttg ggcagtggac gggaccaggt 4260
gccgtggagc agtgggaggc acccatccgg gaggctcggg cctcgcaggg agcccaccgt 4320
agggaggctt gggcatggca ggctgcaagt cctgagccct gccccgcggg gaggtgactg 4380
aggcctggcg acaattcaag tgtggtgagc gccggcaggc cagcagtact gggggacccg 4440
gtgccccctc tgcagctgct ggcccaggtg ctaagcccct cactgcctgg ggccagaggc 4500
accagccggc cgctccgagt gcagggcccg ctgagcccct gcccacccag aactggtgct 4560
ggcccgcgag caacccaggt tcccgcacac gcctctccct ccatacctcc ccgcaagcag 4620
acggagccgg ctccagcctc caccagtcca gagaggggct cccacagtgc agcgctgggc 4680
tgaagggctc ctcaagtgtg gtcagagcag aagctgaggc cgaggaggcg ctgagagcga 4740
gcgaggacca ccagcacgtt gacacctctc aacctcacca caggactggc cacctctctg 4800
ggccctcagg gatgctgctg tctggacccc tgaccagtga cgagttcgca ctcagggcca 4860
ggctggcgct ggaggaggac acttgtttgg ctccaaccct aggtaccatc ctcccagtag 4920
ggatcaggca gggcccacag gcctgcccta gggacaggag tcaaccttgg acccataagg 4980
cactggggcg ggcagagaag gaggaggtgg catgggcagc tgagagccag agaccctgac 5040
cctagtcctt gctctgccat taccccgtgt gaccccgggc ccacccttcc ccacccttcc 5100
ccacccttcc ccaccccggg cttctgtttc ccttctgcca acgagaaggc tgcttcacct 5160
gccccgagtc ctgtcttcct gctctgcctt ctggggctgt ggcccttgct ggcctggagc 5220
cccaaccaag ggcagggact gctgtcctcc acgtctgtcc tcaccgacat aatgggctgg 5280
gctgggcaca caggcagtgc ccaagagttt ctaatgagca tatgattacc tgagtcctgg 5340
gcagaccttc ttagggaaca gcctgggaca gagaaccaca gacactctga ggagccacct 5400
gaggcctctt ttgccagagg accctacagc ctccctggca gcagttccgc cagcatttct 5460
gtaaatgccc tcatgccagg gtgcggcccg gctgtcagca cgagagggac gttggtctgt 5520
cccctggcac cgagtcagtc agaagggtgg ccagggcccc cttgggcccc tccagagaca 5580
atccactgtg gtcacacggc tcggtggcag gaagtgctgt tcctgcagct gtggggacag 5640
ggagtgtgga tgaagccagg ctgggtttgt ctgaagacgg aggccccgaa aggtggcagc 5700
ctggcctata gcagcagcaa ctcttggatt tattggaaag attttcttca cggttctgag 5760
tcttgggggt gttagaggct cagaaccagt ccagccagag ctctgtcatg ggcacgtaga 5820
cccggtccca gggcctttgc tctttgctgt cctcagaggc ctctgcaaag tagaaacagg 5880
cagccttgtg agtcccctcc tgggagcaac caaccctccc tctgagatgc cccggggcca 5940
ggtcagctgt ggtgaaaggt agggatgcag ccagctcagg ggagtggccc agagttcctg 6000
cccacccaag gaggctccca ggaaggtcaa ggcacctgac tcctgggctg cttccctccc 6060
ctcccctccc caggtcagga aggtgggaaa gggctggggt gtctgtgacc ctggcagtca 6120
ctgagaagca gggtggaagc agccccctgc agcacgctgg gtcagtggtc ttaccagatg 6180
gatacgcagc aacttccttt tgaacctttt tattttcctg gcaggaagaa gagggatcca 6240
gcagtgagat caggcaggtt ctgtgttgca cagacaggga aacaggctct gtccacacaa 6300
agtcggtggg gccaggatga ggcccagtct gttcacacat ggctgctgcc tctcagctct 6360
gcacagacgt cctcgctccc ctgggatggc agcttggcct gctggtcttg gggttgagcc 6420
agcctccagc actgcctccc tgccctgctg cctcccactc tgcagtgctc catggctgct 6480
cagttggacc cacgctggag acgttcagtc gaagccccgg gctgtcctta cctcccagtc 6540
tggggtacct gccacctcct gctcagcagg aatggggcta ggtgcttcct cccctgggga 6600
cttcacctgc tctccctcct gggataagac ggcagcctcc tccttggggg cagcagcatt 6660
cagtcctcca ggtctcctgg gggtcgtgac ctgcaggagg aataagaggg cagactgggc 6720
agaaaggcct tcagagcacc tcatcctcct gttctcacac tggggtgtca cagtcctggg 6780
aagttcttcc ttttcagttg agctgtggta accttgtgag tttcctggag ggggcctgcc 6840
actacccttg ggactccctg ccgtgtgtct gggtctaact gagctctgaa aggagagagc 6900
cccagccctg ggccttccag gggaagcctt acctcagagg ttggcttctt cctactcttg 6960
actttgcgtc tctgcagagg gaggtgggag gggtgacaca accctgacac ccacactatg 7020
agtgatgagt agtcctgccc cgactggccc atcctttcca ggtgcagtcc cccttactgt 7080
gtctgccaag ggtgccagca cagccgcccc actccagggg aagaggagtg ccagccctta 7140
cccacctgag tgggcacagt gtagcattta ttcattagcc cccacactgg cctgaccatc 7200
tcccctgtgg gctgcatgac aaggagagag aacaggctga ggtgagagct actgtcaaca 7260
cctaaaccta aaaaatctat aattgggctg ggcagggtgg ctcacgcctg taatcccagc 7320
actttgggag gccgagatgg gtggatcacc tgaggtcaga tgttcgagac cagcctggcc 7380
aacatggtga aaccccgtct ctactaaaaa tacaaaaaat tagctgggcg tggtggtggg 7440
tgcctgtaat cccagctact caggaggctg aggcaggaga attgcttgaa cctgggaggc 7500
agaggttgca gtgagccgag atcacaccat tgcactccag tctgggtgat aagtatgaaa 7560
cgccatctcc aaaacaaaag aaaagcctaa ttccccaaga actgtcagtc tttcacctgt 7620
ctgctagctc ccagggagac cccacttgcc agggctgtct acatttgtcc tgagatctct 7680
tctggtgaga acagcacttt cctcaggaaa gtttgttgaa agtcatcaga tccatgattg 7740
aaaatcgaag ctgcctgtgg tgatggataa cagctggggt taaaaagcag cagctggggc 7800
atgagcggtc cacagtgagt ttttgttgtt tttgtttttt tgggtggggg atggggtctt 7860
gctaggtctc aaactcctgg cctcaagtca tcctcccatt acagccttct gagtcactga 7920
cactacaggt gtgagccacc atgtccagct tgtagtggtt ttgaacagct cttgcccctt 7980
cttgggaatc taggtgccct gcacgtgggt aaggctgtct gcagctgtgc ccatattcag 8040
gaaggccggc aaggccctga gccctcacac gtgactgacc tgaggtgctg tgcagacagc 8100
aggtgacggc taagggaaag ttgagcactg cctagccgag cactgaagcc acgcccggca 8160
cacagagaga gacccactcg gcaaagactt cgcttccagg cacctaagga actctctgac 8220
cagtcattag ctgaccactg ccgtaactga agagcggctt cagtggccac agctcgcagg 8280
gaatggagac attaatgctt agtcagaatt agttcagaaa agtcacccag caaagaaaca 8340
gctccaacag gcaacaacaa caacacatcc ttggcaggga agagaatctg acttccggag 8400
ttgccacatt atcgcccgtg aaatgtccag gttttaacaa attatgagac atggaaagga 8460
aaccgaaagg acgacccaga cacgggaaaa gtcaccaatg ggaccagccc gatgctgcaa 8520
ttgctagaca aagacgttca gtcagctcat ttaaatatgt tcaaagacct aaaacatgct 8580
gcatctgagg ctgcaccggc tggaacctgc tgatctcgga agctaagcat ggtcaggcct 8640
ggctagtact tcaaagggag aaaccacgtg taggcctggt gcagtggctc acacctataa 8700
tcctagcact ctgggaagct gaggcccgtg gattgcttga gcccaggagt ttgagagcag 8760
cttgggaaat gtggtgagac ccccatctct acaaaaaaat ttaaaaaatt agctggctgc 8820
ctatggtccc agcctctcag gatgctgagg taggaggatc acttcagccc aggaagttga 8880
ggctgcagtg agccatgact gcatcactgc actccagctt gggcgacaga gagaccctct 8940
cccaagaaaa agaaaagaac catgtcaaaa gaactaacga aagtgtggga acaatgtctc 9000
accaattaga gaatatcaat aatgggatga accttataaa aaggggctgg gcatggtggc 9060
tcatgcctat aatcccagca ctttgggagg ctgaggcggg catatcatga ggtcaagaga 9120
ttgagaccag cctggccaac atggtgaaac cccgtctcta cttaaaatac aaaaattagc 9180
cgggcgtggt ggcacgtgcc tgtaatccca gctactcggg aggctgaggc aggagaatcg 9240
cttgaacccg agaggcagag attgcagtga gccgagattg caccactgca ctacagcctg 9300
ggtgacagag cgatactcca aaaaacaaaa caaaacaaaa aacaaaaaaa aagtttaaaa 9360
aggaaccaaa taaaaattct ggagttgtag ggtaaaataa atgaaaattc atcccagggg 9420
cccaagagca gattggaaca attggaagaa agagcctgtg actatggaga gaggccacct 9480
gaggtagtcc cctctgagga acaggaacaa gcatgaagag caatgcacag agatccagag 9540
acctggagac gccgtcaagc tttccgacat acacacaatg ggagtcccag gaaagaagac 9600
agggagaaag gagtaaagga atagttgaag aattaatggc tgaaaaacct cccaaatctg 9660
atgaaaaata ttaatccgta catccaaaaa gctcatcaaa ctccaagtag ggtaaactca 9720
aagagatctt cagccatacg catcatcata atcactgtca aaagacagat ttttcttttt 9780
ttagaatttt aaatgtacct tttaatttgc tcctggggca aagagccagg actggtacta 9840
gagcagtgtc tgggatgaga agaatttaat aaaatgggat taggtccaat ggttgggtta 9900
ggggaggcaa cctgctcgga aggatcagcc tcaacctatc catgcagcag ggcctccacc 9960
tgtccctctc cgtagtccca cacctggaac ccagagccat ctgcctcttc ccagatcatg 10020
gccgacagca ctccaccaga ctgcggctgg agcaggcaca ggattcactt attgagggct 10080
gtggcctggc acagatcata gcctataccc agggacagtt gtgtcacttc tgccaccacc 10140
acatccgcct tctgcagcca catcaagtac cactcatgga tgagcccgtc acccccagcg 10200
gacttatcaa ccccgcgtcc agctccacag ccgccacgtg ctcggtgagc actggctcca 10260
agcatggcag ctgccataca atccacctgt agagggcccg gtcctcctgt cctcagtgga 10320
tgatcccata gaagtccaga gctcggcagc tgccctccca caaaagacag gattttgaaa 10380
gcagcaagag agaagagacg tatcaggtag tcacagtggc tcaggcctgt aatcccagca 10440
ctttgggagg cccaggtggg aggatcgctt caccccagga attcaagacc agcctggaca 10500
acttggaaga acccggtctc tacaaaaaat acaaaattag ctgggattgg gtgcggtggc 10560
tcatgcctat aatcccagca ctttgggagc ctgaggtggg tggatcacct gaagtcagga 10620
gttcaagact agcctggcca acatggtgaa accctatctc tactgaaaat ataaaaagct 10680
agacgtggtg gcacacacct gtaatcccag ctacttagga ggctgaggca ggagaattgc 10740
ttgaagccta gaggtgaagg ttgtagtgag ccgagattgc atcattgcac aatggagggg 10800
agccaccagc ctgggcaaca agaggaaatc tccgtctcca aaaaaaaaaa aaaaaaaaaa 10860
aaaaggatta ggctgggtgg tgcctgtagt cccagctact tgggaggcag ggggtccact 10920
tgatgtcgag actgcagtga gccatgatcc tgccactgca ctccggcctg ggcaacagag 10980
tgagaccctg tctaaagaaa aaaaaaataa agcaacatat cctgaacaaa ggatcctcca 11040
taacgttccc accagatttc taatcagaaa catggaggcc agaaagcagt ggaggaggac 11100
aaccctcagg cagcccggga ggatgttgtc acaggctggg gcaagggcct tccggctacc 11160
aactgggagc tctgggaaca gccctgttgc aaacaagaag ccatagcccg gccagagccc 11220
aggaatgtgg gctgggctgg gagcagcctc tggacaggag tggtcccatc caggaaacct 11280
ccggcatggc tgggaagtgg ggtacttggt gccgggtctg tatgtgtgtg tgactggtgt 11340
gtgtgagaga gaatgtgtgc cctaagtgtc agtgtgagtc tgtgtatgtg tgaatattgt 11400
ctttgtgtgg gtgattttct gcgtgtgtaa tcgtgtccct gcaagtgtga acaagtggac 11460
aagtgtctgg gagtggacaa gagatctgtg caccatcagg tgtgtgcata gcgtctgtgc 11520
atgtcaagag tgcaaggtga agtgaaggga ccaggcccat gatgccactc atcatcagga 11580
gctctaaggc cccaggtaag tgccagtgac agataagggt gctgaaggtc actctggagt 11640
gggcaggtgg gggtagggaa agggcaaggc catgttctgg aggaggggtt gtgactacat 11700
tagggtgtat gagcctagct gggaggtgga tggccgggtc cactgaaacc ctggttatcc 11760
cagaaggctt tgcaggcttc aggagcttgg agtggggaga gggggtgact tctccgacca 11820
ggcccctcca ccggcctacc ctgggtaagg gcctggagca ggaagcaggg gcaagaacct 11880
ctggagcagc ccatacccgc cctggcctga ctctgccact ggcagcacag tcaacacagc 11940
aggttcactc acagcagagg gcaaaggcca tcatcagctc cctttataag ggaagggtca 12000
cgcgctcggt gtgctgagag tgtcctgcct ggtcctctgt gcctggtggg gtgggggtgc 12060
caggtgtgtc cagaggagcc catttggtag tgaggcaggt atggggctag aagcactggt 12120
gcccctggcc gtgatagtgg ccatcttcct gctcctggtg gacctgatgc accggcgcca 12180
acgctgggct gcacgctact caccaggccc cctgccactg cccgggctgg gcaacctgct 12240
gcatgtggac ttccagaaca caccatactg cttcgaccag gtgagggagg aggtcctgga 12300
gggcggcaga ggtgctgagg ctcccctacc agaagcaaac atggatggtg ggtgaaacca 12360
caggctggac cagaagccag gctgagaagg ggaagcaggt ttgggggact tcctggagaa 12420
gggcatttat acatggcatg aaggactgga ttttccaaag gccaaggaag agtagggcaa 12480
gggcctggag gtggagctgg acttggcagt gggcatgcaa gcccattggg caacatatgt 12540
tatggagtac aaagtccctt ctgctgacac cagaaggaaa ggccttggga atggaagatg 12600
agttagtcct gagtgccgtt taaatcacga aatcgaggat gaagggggtg cagtgacccg 12660
gttcaaacct tttgcactgt gggtcctcgg gcctcactgc tcaccggcat ggaccatcat 12720
ctgggaatgg gatgctaact ggggcctctc ggcaattttg gtgactcttg caaggtcata 12780
cctgggtgac gcatccaaac tgagttcctc catcacagaa ggtgtgaccc ccacccccgc 12840
cccacgatca ggaggctggg tctcctcctt ccacctgctc actcctggta gccccggggg 12900
tcgtccaagg ttcaaatagg actaggacct gtagtctggg gggatcctgg cttgacaaga 12960
ggccctgacc ctccctctgc agttgcggcg ccgcttcggg gacgtgttca gcctgcagct 13020
ggcctggacg ccggtggtcg tgctcaatgg gctggcggcc gtgcgcgagg cgctggtgac 13080
ccacggcgag gacaccgccg accgcccgcc tgtgcccatc acccagatcc tgggttttgg 13140
gccgcgttcc caaggcaagc agcggtgggg acagagacag atttccgtgg gacccgggtg 13200
ggtgatgacc gtagtccgag ctgggcagag agggcgcggg gtcgtggaca tgaaacaggc 13260
cagcgagtgg ggacagcggg ccaagaaacc acctgcacta gggaggtgtg agcatgggga 13320
cgagggcggg gcttgtgacg agtgggcggg gccactgccg agacctggca ggagcccaat 13380
gggtgaggct ggcgcatttc ccagctggaa tccggtgtcg aagtgggggg cggggaccgc 13440
acctgtgctg taagctcagt gtgggtggcg cggggcccgc ggggtcttcc ctgagtgcaa 13500
aggcggtcag ggtgggcaga gacgaggtgg ggcaaagccc tgccccagcc aagggagcaa 13560
ggtggatgca caaagagtgg gccctgtgac cagctggaca gagccaggga ctgcgggaga 13620
ccagggggag catagggttg gagtgggtgg tggatggtgg ggctaatgcc ttcatggcca 13680
cgcgcacgtg cccgtcccac ccccaggggt gttcctggcg cgctatgggc ccgcgtggcg 13740
cgagcagagg cgcttctccg tctccacctt gcgcaacttg ggcctgggca agaagtcgct 13800
ggagcagtgg gtgaccgagg aggccgcctg cctttgtgcc gccttcgcca accactccgg 13860
tgggtgatgg gcagaagggc acaaagcggg aactgggaag gcgggggacg gggaaggcga 13920
ccccttaccc gcatctccca cccccaggac gcccctttcg ccccaacggt ctcttggaca 13980
aagccgtgag caacgtgatc gcctccctca cctgcgggcg ccgcttcgag tacgacgacc 14040
ctcgcttcct caggctgctg gacctagctc aggagggact gaaggaggag tcgggctttc 14100
tgcgcgaggt gcggagcgag agaccgagga gtctctgcag ggcgagctcc cgagaggtgc 14160
cggggctgga ctggggcctc ggaagagcag gatttgcgta gatgggtttg ggaaaggaca 14220
ttccaggaga ccccactgta agaagggcct ggaggaggag gggacatctc agacatggtc 14280
gtgggagagg tgtgcccggg tcagggggca ccaggagagg ccaaggactc tgtacctcct 14340
atccacgtca gagatttcga ttttaggttt ctcctctggg caaggagaga gggtggaggc 14400
tggcacttgg ggagggactt ggtgaggtca gtggtaagga caggcaggcc ctgggtctac 14460
ctggagatgg ctggggcctg agacttgtcc aggtgaacgc agagcacagg agggattgag 14520
accccgttct gtctggtgta ggtgctgaat gctgtccccg tcctcctgca tatcccagcg 14580
ctggctggca aggtcctacg cttccaaaag gctttcctga cccagctgga tgagctgcta 14640
actgagcaca ggatgacctg ggacccagcc cagccccccc gagacctgac tgaggccttc 14700
ctggcagaga tggagaaggt gagagtggct gccacggtgg ggggcaaggg tggtgggttg 14760
agcgtcccag gaggaatgag gggaggctgg gcaaaaggtt ggaccagtgc atcacccggc 14820
gagccgcatc tgggctgaca ggtgcagaat tggaggtcat ttgggggcta ccccgttctg 14880
tcccgagtat gctctcggcc ctgctcaggc caaggggaac cctgagagca gcttcaatga 14940
tgagaacctg cgcatagtgg tggctgacct gttctctgcc gggatggtga ccacctcgac 15000
cacgctggcc tggggcctcc tgctcatgat cctacatccg gatgtgcagc gtgagcccat 15060
ctgggaaaca gtgcaggggc cgagggagga agggtacagg cgggggccca tgaactttgc 15120
tgggacaccc ggggctccaa gcacaggctt gaccaggatc ctgtaagcct gacctcctcc 15180
aacataggag gcaagaagga gtgtcagggc cggaccccct gggtgctgac ccattgtggg 15240
gacgcatgtc tgtccaggcc gtgtccaaca ggagatcgac gacgtgatag ggcaggtgcg 15300
gcgaccagag atgggtgacc aggctcacat gccctacacc actgccgtga ttcatgaggt 15360
gcagcgcttt ggggacatcg tccccctggg tgtgacccat atgacatccc gtgacatcga 15420
agtacagggc ttccgcatcc ctaaggtagg cctggcgccc tcctcacccc agctcagcac 15480
cagcccctgg tgatagcccc agcatggcta ctgccaggtg ggcccactct aggaaccctg 15540
gccacctagt cctcaatgcc accacactga ctgtccccac ttgggtgggg ggtccagagt 15600
ataggcaggg ctggcctgtc catccagagc ccccgtctag tggggagaca aaccaggacc 15660
tgccagaatg ttggaggacc cagcgcctgc agggagaggg ggcagtgtgg gtgcctctga 15720
gaggtgtgac tgcgccctgc tgtggggtcg gagagggtac tgtggagctt ctcgggcgca 15780
ggactagttg acagagtcca gctgtgtgcc aggcagtgtg tgtcccccgt gtgtttggtg 15840
gcaggggtcc cagcatccta gagtccagtc cccactctca ccctgcatct cctgcccagg 15900
gaacgacact catcaccaac ctgtcatcgg tgctgaagga tgaggccgtc tgggagaagc 15960
ccttccgctt ccaccccgaa cacttcctgg atgcccaggg ccactttgtg aagccggagg 16020
ccttcctgcc tttctcagca ggtgcctgtg gggagcccgg ctccctgtcc ccttccgtgg 16080
agtcttgcag gggtatcacc caggagccag gctcactgac gcccctcccc tccccacagg 16140
ccgccgtgca tgcctcgggg agcccctggc ccgcatggag ctcttcctct tcttcacctc 16200
cctgctgcag cacttcagct tctcggtgcc cactggacag ccccggccca gccaccatgg 16260
tgtctttgct ttcctggtga gcccatcccc ctatgagctt tgtgctgtgc cccgctagaa 16320
tggggtacct agtccccagc ctgctcccta gccagaggct ctaatgtaca ataaagcaat 16380
gtggtagttc caatcgagct agagctcgct gatcagcctc gactgtgcct tctagttgcc 16440
agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 16500
ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 16560
ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 16620
atgctgggga ataacttcgt ataatgtatg ctatacgaag ttattctacc gggtagggga 16680
ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg 16740
ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc 16800
tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc 16860
ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag 16920
tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg 16980
ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg 17040
gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc 17100
ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc 17160
gggcctttcg acctgcagcc tgttgacaat taatcatcgg catagtatat cggcatagta 17220
taatacgaca aggtgaggaa ctaaaccatg ggatcggcca ttgaacaaga tggattgcac 17280
gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 17340
atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 17400
gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 17460
tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 17520
agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 17580
cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 17640
gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 17700
gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 17760
gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt cgtgacccat 17820
ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 17880
tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 17940
gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 18000
cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aggggatcaa 18060
ttctctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 18120
tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 18180
taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 18240
gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 18300
gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgactag agcttgcgga 18360
acccttcata acttcgtata atgtatgcta tacgaagtta tttagccacc tgcctttgct 18420
gtcttttctc tgttcctgtc cccccccccc cacctcaatt aggtcccaga gtgcctccaa 18480
tccccaaact ctctctagtg cccatttgcc caccactccc gccactgtct gcatttgcct 18540
tgagagcgtg gtccaaagcc cagcaggtca ttgaaatgca gcggtggccg gtcagcagtg 18600
tcctcgctgt gtttcaccag tgcctctcgc agggccgtca gcccatttag tacaaccact 18660
gactcagagg ccagctgtag gctgaacagg tccccatagc gaccccgcag ctgtaggcaa 18720
ggagaatgca ttacgggcaa gcctggattc ctgcacttag ctgtaccgga taggacttgg 18780
caggggagac ccagggtgag caccacagaa ggttggcatc tgctactatt tgtctgggga 18840
accaccttgg actctgtcaa tcacctcatc atgaccttgc tagtgtctgt cacacatctg 18900
ccagctggtc ccactagcat ccccatgtct agatgaagaa actaaggctc acagatgtaa 18960
gggagtctga gaatctgaaa cagcaaaagg atggaggccg gcatttccca tctctcagct 19020
ttgatttcct gacagaaaca cctgtttagg atcatggcct ctccaccccc cctttttttt 19080
tttttttata aaaagactta atgtatatga atacattggt gctatctcca gacacatcag 19140
aagagggaat tgaattccat tgcagatggt tatgagtcac catgtggttg ctgggaattg 19200
aactcaggac ctctggaagt gcttaacctc tgggccatct ttccagtgcc cccttccaca 19260
ttcttaaaac atttccttct ggtggaatga aaaggaacag gaagcctata tgacttgatt 19320
tccctttgtg tgtgtgtgtg tgtgtataat ttttgtctta gcatgtagcc agggctggct 19380
tggaattttc tacgtagatc aagctggtgc tgaattcaca gagatctgct ggcttttgcc 19440
tcctctgaat gctgggatta aaggtctcca cgactgcatt gggctttttc cttttaaaat 19500
gttagatttc tttctttctt tttctttttc tttttctttt tctttttctt ttcttttttg 19560
aaacagtttc actgtgtagc gtgggctgtc ctggaactca gtatgttaga tgagactggt 19620
ctagagctca aagaaatatg cccgcctctg cctcttaagt gctggggtta gaggcacaca 19680
ccttcacagc tggccttgaa gtctttggtt tagagatggt ttaagatgga atttataacc 19740
cagctgcttt agcctcccaa ttacaggcat gctggaatcg gaaagcaacc ccagcactac 19800
cgctaccatc ctgttaactg catctcaaac tggctgctag gatccatccc gcctcctagc 19860
tgttgccttt ggaaaatcca gcatgacctt gggtcctgca ccactctcct cacgggggag 19920
cctcttaacc tgcgttctgg gggggggggg gcagctccct gtcccgcatg cagttttacc 19980
accacccact tgtggtcacc atggcttctc ctgccctcca aaaattccct taccttttgg 20040
aagcctgctg gcatattctg gaagtccatg tgcagcaggt tcccaagcac aggccacggc 20100
atagggcctg gagggtagtg ggcagtccag cgctggcgcc agtgcatcag gttcaccaga 20160
atcaggaaga tgaccgtgaa tatggctatg ggccacagtt cagccccggt cggcagtctc 20220
atggctgccc ggctgctctt tgggtttctt tggaaacact tctcaatcct aactccagct 20280
acctaggacg aaggaataac aaacagtatt atcagaccct gggaactcac ctggcctggc 20340
ctggactctt gtaaaggaga tgctgctgcc ctttatcctc tgccttaagc caagctgctg 20400
cattgaccac gtagagggca aggaagggtc aaggcaggcc agggacaatg gccttagaag 20460
tccctccctc taagtcctgt gtgtgtgtgt gtgtgtgtgt gtgttttgca tctcaacatg 20520
tcttcaggag gaatgataga aaattgtagg cccgggcaat agctactgat ggtgtaggga 20580
attagaaagt catgattaag ggtttgcctc acagcaggag attcagtact tggatgcctg 20640
tgaccttatc tagagccagg ttctccacag aatcaaggaa cacaggtagt atgaactgaa 20700
gcatcagata agcagcatag cttggctggc ctctggtgcg gtgtctccta ctcccacagg 20760
cccaatttca ctttgccaca cgcttctcgt caaacaggat ggctaaaagc cagctcagac 20820
gtgtcccata cccttaaact gcgagcggct ggcaaatgct agcaattgca cgctgccgca 20880
gaaggctccc aggcccacta gctcatttga tcatcaacgt ttaaagagaa gactctaggc 20940
ttttcctagt gacccagacc ggtcactctc tactccagac ccactagaat gcagatccta 21000
gctctccaaa ccatctgcac aagcctgtga gtcagcaaga ctaacaggag cttctcatac 21060
tactctaacc taaaaacttc ccagcctctg agccagacta tggctgtctg cagccaagaa 21120
acattccatt ggcgcgttat gaaaacgcct ttgtttgtgg gcgaggaccc aaacaggaaa 21180
aaaagcatcc tttgttgagt tacaatcaac atcgctgagt ggaaatcaca gtgccatggg 21240
gggagagctc gtctccctcc ccatcctgtc cccacccctt ctctcttgtt ttccagacag 21300
ctgcaatatt tggcaacttt gaagaagttg gctgctttct tagctgggag aaccacgtct 21360
gtatggctgt ctctgtggat gataagtagg tacagagtat agatcctcta gagtcgagca 21420
gtgtggtttt caagaggaag caaaaagcct ctccacccag gcctggaatg tttccaccca 21480
atgtcgagca gtgtggtttt gcaagaggaa gcaaaaagcc tctccaccca ggcctggaat 21540
gtttccaccc aatgtcgagc aaaccccgcc cagcgtcttg tcattggcga attcgaacac 21600
gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg tgacgcgtgt 21660
ggcctcgaac accgagcgac cctgcagcga cccgcttaac agcgtcaaca gcgtgccgca 21720
gatcttggtg gcgtgaaact cccgcacctc ttcggccagc gccttgtaga agcgcgtatg 21780
gcttcgtacc ccggccatca gcacgcgtct gcgttcgacc aggctgcgcg ttctcgcggc 21840
catagcaacc gacgtacggc gttgcgccct cgccggcagc aagaagccac ggaagtccgc 21900
ccggagcaga aaatgcccac gctactgcgg gtttatatag acggtcccca cgggatgggg 21960
aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat cgtctacgta 22020
cccgagccga tgacttactg gcgggtgctg ggggcttccg agacaatcgc gaacatctac 22080
accacacaac accgccttga ccagggtgag atatcggccg gggacgcggc ggtggtaatg 22140
acaagcgccc agataacaat gggcatgcct tatgccgtga ccgacgccgt tctggctcct 22200
catatcgggg gggaggctgg gagctcacat gccccgcccc cggccctcac cctcatcttc 22260
gaccgccatc ccatcgccgc cctcctgtgc tacccggccg cgcgatacct tatgggcagc 22320
atgacccccc aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt gcccggcaca 22380
aacatcgtgt tgggggccct tccggaggac agacacatcg accgcctggc caaacgccag 22440
cgccccggcg agcggcttga cctggctatg ctggccgcga ttcgccgcgt ttacgggctg 22500
cttgccaata cggtgcggta tctgcagggc ggcgggtcgt ggcgggagga ttggggacag 22560
ctttcgggga cggccgtgcc gccccagggt gccgagcccc agagcaacgc gggcccacga 22620
ccccatatcg gggacacgtt atttaccctg tttcgggccc ccgagttgct ggcccccaac 22680
ggcgacctgt acaacgtgtt tgcctgggcc ttggacgtct tggccaaacg cctccgtccc 22740
atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg gctgccggga cgccctgctg 22800
caacttacct ccgggatgat ccagacccac gtcaccaccc caggctccat accgacgatc 22860
tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg ctaactgaaa cacggaagga 22920
gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg 22980
gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata 23040
ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc 23100
ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca 23160
tagccacggg ccccgtgggt tagggacggg gtcccccatg gggaatggtt tatggttcgt 23220
gggggttatt attttgggcg ttgcgtgggg tcagtccacg actggactga gcagacagac 23280
ccatggtttt tggatggcct gggcatggac cgcatgtact ggcgcgacac gaacaccggg 23340
cgtctgtggc tgccaaacac ccccgacccc caaaaaccac cgcgcggatt tctggcgccg 23400
ccggacgaac taaacctgac tacggcatct ctgccccttc ttcgctggta cgaggagcgc 23460
ttttgttttg tattggtcac cacggccgag tttcctcgac cgatgccctt gagagccttc 23520
aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact 23580
gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc 23640
gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc 23700
ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag 23760
caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg 23820
acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg catcgggatg 23880
cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa 23940
ggatcgctcg cggctcttac cagcctaact tcgatcactg gaccgctgat cgtcacggcg 24000
atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta 24060
taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga 24120
atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat 24180
tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg 24240
ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc 24300
gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt 24360
agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg 24420
cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc 24480
ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac 24540
cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc 24600
tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg 24660
catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta 24720
cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg 24780
cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg 24840
agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc 24900
tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 24960
tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 25020
gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata 25080
gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca 25140
tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 25200
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 25260
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 25320
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 25380
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 25440
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 25500
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 25560
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 25620
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 25680
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 25740
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 25800
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 25860
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 25920
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 25980
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 26040
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 26100
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 26160
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 26220
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 26280
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 26340
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 26400
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat 26460
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 26520
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 26580
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 26640
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 26700
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga 26760
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 26820
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 26880
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 26940
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 27000
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 27060
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 27120
gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 27180
cacgaggccc tttcgtcttc aagaa 27205
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
ggcactctgg gacctaattg agg 23
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gttcaaacag agcctatatg ggg 23

Claims (6)

1. A method of constructing a humanized CYP2D6 x 10 transgenic mouse model, comprising the steps of: knocking in an expression frame of human CYP2D6 x 10 (hYP 2D6 x 10) at a fixed point between mouse Cyp2D22 gene exon4-5 by utilizing a homologous recombination principle, obtaining a positive clone, knocking out a Cyp2D gene cluster, and obtaining an hYP 2D6 x 10 gene targeting vector; obtaining Cyp2dKO positive ES cells after targeting, injecting the cells into blastocysts of a mouse through amplification, and then transferring the blastocysts into a surrogate mouse for breeding to obtain a chimeric mouse;
the nucleotide sequence of hYP 2D6 x 10 is sequence 1 in the list;
the sequence of the hYP 2D 6-10 gene targeting vector is shown as sequence 2 in the list.
2. The method of claim 1, wherein the hYP 2D6 x 10 gene targeting vector was verified by EcoRI restriction endonuclease digestion and separated by agarose gel electrophoresis to have 10478bp, 8673bp, 5617bp, 2177bp, 260bp5 bands.
3. The method according to claim 1, wherein the positive ES cells obtained after targeting hYP 2D6 x 10 gene are identified by PCR, the 3.6kb fragment should be amplified from the 5' arm homologous recombination positive clone, and the product should be absent from the negative clone; the 3' arm homologous recombination positive clone should amplify 6.0kb fragment, and the negative clone should have no product.
4. The method of claim 1, wherein the Cyp2d gene cluster is knocked out using criprpr/Cas 9 technology, Cyp2dKO positive clones are identified using PCR, a 2.2kb fragment should be amplified from KO positive clones, and no product should be obtained from negative clones.
5. The method of claim 4, wherein the criprpr/Cas 9 technique uses sgRNA sequences as listed as sequence 3 and sequence 4.
6. The method of claim 1, wherein the surrogate mouse is a C57BL/6J mouse, chimeric mice of generation F0 are obtained after birth, mice of generation F0 are crossed, and transgenic mice heterozygous or homozygous for hCYP2D6 x 10 are obtained from mice of generation F1.
CN202011499244.0A 2020-12-17 2020-12-17 Construction method of human CYP2D6 x 10 transgenic mouse model Pending CN112626119A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011499244.0A CN112626119A (en) 2020-12-17 2020-12-17 Construction method of human CYP2D6 x 10 transgenic mouse model

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011499244.0A CN112626119A (en) 2020-12-17 2020-12-17 Construction method of human CYP2D6 x 10 transgenic mouse model

Publications (1)

Publication Number Publication Date
CN112626119A true CN112626119A (en) 2021-04-09

Family

ID=75316621

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011499244.0A Pending CN112626119A (en) 2020-12-17 2020-12-17 Construction method of human CYP2D6 x 10 transgenic mouse model

Country Status (1)

Country Link
CN (1) CN112626119A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862299A (en) * 2021-06-02 2021-12-31 上海南方模式生物科技股份有限公司 Vector for homologous recombination and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109197781A (en) * 2018-09-14 2019-01-15 徐州医科大学 The construction method of AURKA-CKO1-N conditional gene knockout mouse model
CN114736926A (en) * 2022-05-19 2022-07-12 苏州药诺泰克新药研发有限公司 Heg1 gene point mutation mouse model, construction method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109197781A (en) * 2018-09-14 2019-01-15 徐州医科大学 The construction method of AURKA-CKO1-N conditional gene knockout mouse model
CN114736926A (en) * 2022-05-19 2022-07-12 苏州药诺泰克新药研发有限公司 Heg1 gene point mutation mouse model, construction method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NICO SCHEER等: "Modeling human cytochrome P450 2D6 metabolism and drug-drug interaction by a novel panel of knockout and humanized mouse lines", 《MOL PHARMACOL》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862299A (en) * 2021-06-02 2021-12-31 上海南方模式生物科技股份有限公司 Vector for homologous recombination and application thereof
CN113862299B (en) * 2021-06-02 2023-11-28 上海南方模式生物科技股份有限公司 Vector for homologous recombination and application thereof

Similar Documents

Publication Publication Date Title
CN108070662B (en) Genetic marker for determining pork quality traits and application thereof
JP2023115348A (en) Viral vectors encoding recombinant fviii variants with increased expression for gene therapy of hemophilia a
CN106544361B (en) Mammalian cell expression vector, expression system, preparation method and application
CN112481271B (en) Transcription factor C/EBPZ for regulating and controlling formation of adipocytes and application thereof
JP2024012440A (en) Compositions and methods for delivering nucleic acids to cochlear and vestibular cells
CN108913718A (en) A kind of preparation method and application of the CAR-T cell of targeting EGFR v III
KR20180070700A (en) A viral vector encoding a recombinant FVIII variant with increased expression for gene therapy of type A haemophilia
CN114540424A (en) IGFBP7 muscle tissue specific knockout mouse animal model and construction method thereof
CN108103088B (en) Optimized gene of recombinant GLP-1 analogue Fc fusion protein and application thereof
CN112608940B (en) Construction method and application of animal model of congenital cataract disease
CN113943737A (en) Application of chicken CTGF gene in inhibiting differentiation of chicken preadipocytes
CN112626119A (en) Construction method of human CYP2D6 x 10 transgenic mouse model
CN112449640A (en) Gene therapy for hemophilia A using viral vectors encoding recombinant FVIII variants with increased expression
CN111100874B (en) Targeting vector, method for integrating exogenous gene into mouse DC-SIGN exon 7 locus to construct BAC clone and application
KR101876488B1 (en) A transformant comprising MYH3 gene and use thereof
CN109022363A (en) A kind of CD-133-CAR-T system constituting method based on PiggyBac carrier
US20230201306A1 (en) Fibroblast growth factor 21 (FGF21) gene therapy for central nervous system disorders
CN114150001A (en) Construction method of CRISPR/Cas9 vector for toxoplasma gondii gene editing
KR102247462B1 (en) Recombinant Lactic acid bacteria having enhanced Productivity for Riboflavin
CN111663187A (en) Staphylococcus aureus transposon sequencing library and construction method
KR20170112017A (en) Genetically modified bacterial strain for the detection of estrogenic compounds, and detection method for estrogenic compounds using the strain
KR101765256B1 (en) High-Copy Number Shuttle Vector for Expression in Leuconostoc sp.
CN110819589B (en) Method for enhancing immune effector cell function
RU2726541C1 (en) Recombinant human breast cancer cell line brcch4e-134, expressing human prostate-specific membrane antigen
KR102109820B1 (en) Mutant estrogen receptor protein having improved binding capacity for Bisphenol A and bacterial strain using the same

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20210409