CN110863007A - Homologous recombinant vector for mammary gland specific expression of antibacterial peptide Cecropin gene and application - Google Patents
Homologous recombinant vector for mammary gland specific expression of antibacterial peptide Cecropin gene and application Download PDFInfo
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- CN110863007A CN110863007A CN201910954117.6A CN201910954117A CN110863007A CN 110863007 A CN110863007 A CN 110863007A CN 201910954117 A CN201910954117 A CN 201910954117A CN 110863007 A CN110863007 A CN 110863007A
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Abstract
The invention provides a homologous recombination vector for specifically expressing an antibacterial peptide Cecropin gene in mammary gland and application, wherein the homologous recombination vector takes a eukaryotic expression vector as a skeleton vector, and at least comprises a positive screening element, a negative screening marker gene, an antibacterial peptide Cecropin gene expression box, and a homologous left arm and a homologous right arm of a rosa26 gene; wherein the positive screening element comprises a recombinase recognition sequence-positive screening marker gene-recombinase recognition sequence which are connected in sequence. The homologous recombination vector is used for transfecting mammalian somatic cells, and the antibacterial peptide Cecropin gene is integrated into the first intron of the rosa26 gene of the positive cell at a fixed point. The method successfully obtains the transgenic pig cell line of the mammary gland specificity expression antibacterial peptide Cecropin, and lays a foundation for preparing the diarrhea-resistant transgenic pig by a transgenic technology.
Description
Technical Field
The invention belongs to the field of animal genetic engineering, and particularly relates to a homologous recombinant vector for mammary gland specific expression of an antibacterial peptide Cecropin gene and application thereof.
Background
Antimicrobial peptides (Cecropin) were first discovered in the last 70 years and were soon isolated. In recent years, the antibacterial peptide is found to have a special action mechanism different from that of the traditional antibiotics, and has the capabilities of inhibiting the growth of viruses, parasites and tumor cells and killing harmful hospitals. Has become a new class of antibacterial, antiviral, antiparasitic and anticancer drugs with great development potential. The molecular weight of the antibacterial peptide is about 4kD and consists of more than 30 amino acid residues. Has the advantages of amphipathy of N-terminal hydrophile and C-terminal hydrophobic hydrolipide, good thermal stability and the like.
Traditional antibiotics destroy or inhibit certain biological processes by binding to specific receptors of pathogens to achieve bacteriostatic or bactericidal effects. When the antibiotic target is mutated, the antibacterial effect is weakened or even eliminated. The antibacterial peptide mainly acts on bacterial cell membranes to destroy the integrity of bacteria and generate a perforation phenomenon, so that the cell contents are overflowed and die. Almost all antibacterial peptides are cationic or amphiphilic and have strong affinity to Lipopolysaccharide (LPS), and LPS is used as a main component of gram-negative bacteria (G-), and the antibacterial peptides can damage the outer cell membrane by substituting cations combined with LPS so as to destroy bacteria; for gram-positive bacteria (G +), the antibacterial peptide can be combined with muramic acid, teichoic acid and amino acid carboxyl with negative charges on the surface of the cell membrane, so that the peptidoglycan layer is damaged to form a transmembrane channel, and intracellular substances are greatly flowed out to cause cell death. Antibacterial peptides have similar effects on viruses, parasites, and tumor cells, but the mechanism is not yet clear and needs to be further studied.
As a big pig raising country, the pig disease epidemic situation seriously restricts the expansion and development of pig raising enterprises. Piglet diarrhea is one of the most serious piglet disease groups at present and is also an important factor causing piglet death. In recent years, the diarrhea of piglets in China is very common, and the annual average morbidity of piglets below 30kg is reported to be 46.5%, the mortality rate is reported to be 10.3%, and great economic loss is brought to the pig raising industry. The infectious factors causing piglet diarrhea mainly refer to infectious pathogens, wherein the viruses mainly comprise porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus and rotavirus, and the bacteria mainly comprise escherichia coli, salmonella, clostridium welchii and the like; the bacteria mainly comprise salmonella, escherichia coli, shigella, staphylococcus aureus, campylobacter and the like. However, the application of the pig vaccines and antibiotics in China still has many problems, such as large side effect, non-ideal immune effect, appearance of super bacteria and the like. Based on the biological function of the antibacterial peptide, the anti-diarrhea capability of the piglets can be enhanced by a transgenic means.
Disclosure of Invention
The invention aims to provide a homologous recombinant vector for specifically expressing an antibacterial peptide Cecropin gene in mammary gland and application thereof.
The invention has the following conception: the antibacterial peptide Cecropin gene is transferred into pig fibroblasts by a genetic engineering means (by constructing a homologous recombinant vector of the mammary gland specificity expression antibacterial peptide Cecropin gene), and is specifically expressed in mammary glands, so that the content of the antibacterial peptide Cecropin in milk is increased, and the aim of reducing the diarrhea incidence of piglets is fulfilled for the piglets.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a homologous recombination vector for specifically expressing an antibacterial peptide Cecropin gene in a mammary gland, wherein the homologous recombination vector takes a eukaryotic expression vector as a skeleton vector, and at least comprises a positive screening element, a negative screening marker gene, an antibacterial peptide Cecropin gene expression cassette and a homologous left arm and a homologous right arm of a rosa26 gene.
Preferably, the homologous recombination vector is a vector BAC as a backbone vector.
Wherein the positive screening element comprises a recombinase recognition sequence-positive screening marker gene-recombinase recognition sequence which are connected in sequence.
One end of the positive screening element is connected with the homologous left arm, and the other end of the positive screening element is connected with the antibacterial peptide Cecropin gene expression cassette; the other end of the antibacterial peptide Cecropin gene expression box is connected with the homologous right arm, and the outer side of the homologous right arm is connected with a negative screening marker gene.
Preferably, the positive selection marker gene includes, but is not limited to, a gene encoding neomycin phosphotransferase (Neo), hygromycin B phosphotransferase, hypoxanthine phosphoribosyl transferase, or puromycin acetyltransferase.
Preferably, the negative selection marker gene includes, but is not limited to, a gene encoding diphtheria toxin or thymidine (dTA).
The recombinase gene of the invention includes a gene encoding Cre enzyme or Flp enzyme.
Preferably, the positive screening element in the homologous recombination vector is Loxp-OCT4 promoter-Cre-PGK-Neo-Loxp. Wherein, the Cre expression frame is regulated and controlled by an embryonic stage specific promoter OCT 4. The neomycin Neo is used as a positive screening marker, and the dTA is used as a negative screening marker. The LoxP site is located upstream of the Cre expression cassette and downstream of the Neo expression cassette.
Preferably, the antibacterial peptide Cecropin gene expression box is a human lactoferrin hLF promoter-antibacterial peptide Cecropin gene-hLF terminator.
More preferably, the homologous recombination vector comprises the following elements linked in sequence: homologous left arm-Loxp-OCT 4 promoter-Cre-PGK-Neo-Loxp-human lactoferrin hLF promoter-antibacterial peptide Cecropin gene-hLF terminator-homologous right arm-PGK-dTA.
Wherein the homologous left arm is the homologous left arm from the pig rosa26 gene, preferably at least 1.1kb sequence from intron 1 of the pig rosa26 gene, and the nucleotide sequence is shown as SEQ ID NO. 1. The homologous right arm is the homologous right arm from the pig rosa26 gene, preferably a segment of at least 6.0kb sequence from intron 1 of the pig rosa26 gene, and the nucleotide sequence is shown as SEQ ID NO. 2.
The nucleotide sequence of the antibacterial peptide Cecropin gene is shown in SEQ ID NO. 3.
In one embodiment of the invention, the homologous recombinant vector for specifically expressing the Cecropin gene is pHS-AVC-YW077Cecropin (figure 1), wherein O represents a specific promoter of the porcine early embryonic development transcription factor OCT 4; c represents a gene encoding recombinase Cre; n represents a commonly used drug screening marker gene Neo of eukaryotic cells; cec represents an antibacterial peptide Cecropin gene; r represents the insertion site rosa26 gene.
The whole length of the nucleotide sequence of the homologous recombinant vector pHS-AVC-YW077cecropin is shown as SEQ ID NO. 4.
In a second aspect, the invention provides a cell line specifically expressing an antibacterial peptide Cecropin gene, and a target-positive cell clone obtained by transfecting a mammalian somatic cell with the homologous recombination vector is the cell line specifically expressing the antibacterial peptide Cecropin gene.
More specifically, the invention provides a transgenic pig fetal fibroblast line specifically expressing the Cecropin, and the homologous recombination vector is transfected into a pig cell line with the same DNA source of a homologous arm thereof to obtain the pig cell line identified as a fixed-point integration antibacterial peptide Cecropin gene.
In a third aspect, the invention provides the use of the homologous recombination vector or the cell line in the preparation of a transgenic animal specifically expressing the Cecropin in mammary gland, wherein the animal comprises a pig.
In the application, the homologous recombination vector is transfected into a pig cell line with the same DNA source of the homologous arm.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the homologous recombinant vector of the mammary gland specificity expression antibacterial peptide Cecropin gene is constructed for the first time, and the transgenic pig cell line of the mammary gland specificity expression antibacterial peptide Cecropin is successfully obtained, so that a foundation is laid for preparing the diarrhea-resistant transgenic pig by using a transgenic technology.
Drawings
FIG. 1 is a plasmid map of the homologous recombinant vector pHS-AVC-YW077cecropin of the present invention.
FIG. 2 shows the results of the identification of the homologous recombination between the short arm (A) and the long arm (B) in example 2 of the present invention. Wherein 1300bp is a short-arm positive fragment. 8000bp is a long-arm positive fragment.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 construction of homologous recombinant vector for specifically expressing Cecropin Gene
1. Construction of intermediate vector pHS-AVC-YW056
1) Homologous Left arm was amplified from pBC1-hLZ-GFP-Neo (Jia et al, 2011) constructed earlier in this laboratory with the following amplification primers:
F:CTCTTCTCTTCCTCCCACAGGGCCTCGTTTGTTACGCTGGAAGG
R:AAGCATAAAAACGCACCAATTGTCATAACTTCGTATAATGTATGCTATACGAACGGTAGCTTAGGTGTTGCCTGGACTC
2) enzyme digestion Seq1-XhoI-Seq2 vector
The Seq1-XhoI-Seq2 backbone vector was digested with XhoI at 37 ℃ for 3h and then heat inactivated at 80 ℃ for 20 min.
3) Establishment of recombination reactions
And (3) carrying out assembly reaction on the PCR fragment and the vector enzyme digestion fragment according to the following table:
| reagent | Volume of |
| 2×EasycloneAssembling reagent | 10ul |
| Left arm | 50ng |
| Seq1-XhoI-Seq2 backbone vector | 50ng |
| ddH2O | Supplementing to 20ul |
And blowing and stirring the mixed system, putting the mixed system into a metal bath for reaction, and keeping the constant temperature at 50 ℃ for reaction for 60min to perform subsequent conversion experiments.
2. Construction of intermediate vector pHS-AVC-YW059
1) The vector pBC1-hLZ-GFP-Neo (Jia et al, 2011) constructed in the early stage of the laboratory is used as a template for amplifying OCT4-Cre-PGK-Neo (two-stage amplification)
F1:TCGACGTTAACGCTAGTGATCTAGAGCTAGGCGCGCCTCCG
R1:CAGGCCAGGTATCTCTGACCAGAGTCATCCTAAAATACAC
F2:GGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTG
R2:GCACCTGAGGAGTGAATTCATCAGAAGAACTCGTCAAGAAGG
2)2) Synthesis of Seq1-XhoI-Seq2 sequences:
GGTTTACCGAGCTCTTATTGGTTTTCAAACTTCATTGACTGTGCC CTCGAGGGTGCGTTTTTATGCTTGTAGTATTGTATAATGTTTTTAAGATCC
enzyme digestion Seq2-GTW-Seq3 vector
The Seq2-GTW-Seq3 backbone vector was digested with EcoRV/MluI at 37 ℃ for 3h, followed by heat inactivation at 80 ℃ for 20 min.
3) Establishment of recombination reactions
And carrying out assembly reaction on the PCR fragment and the vector enzyme digestion fragment according to an easy clone assembly reagent. And blowing and stirring the mixed system, putting the mixed system into a metal bath for reaction, and keeping the constant temperature at 50 ℃ for reaction for 60min to perform subsequent conversion experiments.
3. Construction of the intermediate vector pHS-AVC-YW063
1) Loxp-HLF (amplified in two stages) was amplified using pBC1-hLZ-GFP-Neo (Jia et al, 2011) vector constructed earlier in this laboratory as template
F1:AAAAGTTGGCTCCGAATTTCATAACTTCGTATAGCATACATTATACGAACGGTAGACATTCCGTCATGAACACATGATGTGGAGC
R1:TAGGTCACCTGAACACCTTTCTTGG
F2:AAAGGTGTTCAGGTGACCTATTATTTCCC
R2:GTCGAATTATGATCAGTTACGTCTGCGGTCTGGAGGCGACTTG
2) Restriction enzyme pENTR-L4-MCS2-R1 vector
The pENTR-L4-MCS2-R1 backbone vector was digested with XhoI/XmaI at 37 ℃ for 3h, followed by heat inactivation at 80 ℃ for 20 min.
3) Establishment of recombination reactions
And carrying out assembly reaction on the PCR fragment and the vector enzyme digestion fragment according to an easy clone assembly reagent. And blowing and stirring the mixed system, putting the mixed system into a metal bath for reaction, and keeping the constant temperature at 50 ℃ for reaction for 60min to perform subsequent conversion experiments.
4. Construction of intermediate vector pHS-AVC-YW066
pHS-AVC-YW063, pENTR-L1-Cecropin-L2 (gene synthesis) and Seq3-ccdB-Seq4 (skeleton vector) are respectively diluted into 20nM (10ul system), 0.5ul of LR enzyme and 0.5ul of ddH are respectively added into 0.5ul2And O, reacting at room temperature for 3 hours.
5. Construction of intermediate vector pHS-AVC-YW058
1) Homologous Right arm was amplified from pBC1-hLZ-GFP-Neo (Jia et al, 2011) provided earlier in the laboratory with the following amplification primers:
F1:TCTTCCTCCCACAGGGCCTCGAGACGTTAGTAACTGAGCTCAGTTGCCGGTAGACATTCCGTCATGAACACATGATGTGGAGC
R1:ATTACTGCTTTTGCAAGCAACCGATCACTAGGGACAGTACTGATAGTCAGCCTC
2) enzyme digestion Seq4-GTW-Seq5 vector
The Seq4-GTW-Seq5 backbone vector was digested with EcoRV/MfeI at 37 ℃ for 3h, followed by heat inactivation at 80 ℃ for 20 min.
3) Establishment of recombination reactions
And carrying out assembly reaction on the PCR fragment and the vector enzyme digestion fragment according to an easy clone assembly reagent. And blowing and stirring the mixed system, putting the mixed system into a metal bath for reaction, and keeping the constant temperature at 50 ℃ for reaction for 60min to perform subsequent conversion experiments.
6. Construction of intermediate vector pHS-AVC-YW055
1) pENTR-L4-mPGK-R (pHS-AVC-YW053), pENTR-L1-dTA-L2(pHS-AVC-YW054), Seq5-GTW-Seq6 (backbone vector) were diluted to 20nM (10ul system), respectively.
2) The LR reaction was carried out according to the reaction system shown in the following table, and the reaction was carried out at room temperature for 3 hours.
| Reagent | Volume of |
| LR mixed solution | 0.5ul |
| mPGK | 0.5ul |
| dTA | 0.5ul |
| Skeleton carrier | 0.5ul |
| ddH2O | Supplementing to 2.5ul |
3) After the reaction was completed, a conversion experiment was performed.
7. Assembly of multiple component carriers
Assembling a homologous left arm-Loxp-OCT 4 promoter-Cre-PGK-Neo-Loxp-human lactoferrin hLF promoter-antibacterial peptide Cecropin gene-hLF terminator-homologous right arm-PGK-dTA, namely a homologous recombinant vector pHS-AVC-YW 077.
The construction method comprises the following steps:
1) preparation of linearized fragments
① pHS-AVC-YW056/059/066/058/055 calculated 42fmol per vector was placed in the same centrifuge tube and digested with I-SceI for 3 hours (10ul system) and then heat inactivated at 80 ℃ for 20 min.
② the pZ306 vector (42fmol) was digested with PacI for 3 hours (10ul system) and then heat-inactivated at 80 ℃ for 20 min.
③ the pKAn-6-X vector (42fmol) was digested with XbaI/XhoI for 3 hours (10ul system) and then heat-inactivated at 80 ℃ for 20 min.
2) Establishment of recombination reaction System
① the assembled mixed solution of 1.33 x gibson assembly is taken out from a refrigerator at-20 deg.C and stored on ice, and if icing occurs, the ice is placed on ice for melting.
| Reagent | Volume of |
| IOS buffer 5X | 400ul |
| T5exonuclease*(10U/ul) | 0.8ul |
| Phusion polymerase(2U/ul) | 25ul |
| Taq ligase(40U/ui) | 200ul |
| ddH2O | 375ul |
| Total | 1ml |
② mu.l of 1.33 Xgibson assembly is taken out and put into a PCR tube, solutions containing the ligated fragments are added in order at an equimolar ratio to a total volume of 20. mu.l, and if the total volume is less than 20. mu.l after all the fragment solutions are added, ddH is used2The amount of O was made up to 20. mu.l. Reagents were added to 0.2ml centrifuge tubes according to the following table:
| reagent | Volume of |
| 1.33 Xgibson assembly mixture | 15ul |
| Assembling segments | 1.67ul |
| Linearized pZ306 | 1.67ul |
| pKan-6-X | 1.67ul |
③ and blowing and stirring the mixed system, and keeping the constant temperature at 50 ℃ for reaction for 60 min.
④ if the conversion cannot be carried out immediately after the reaction, it should be stored at-20 ℃.
⑤, after the sequence verification, the amplification culture is carried out, and the endotoxin is removed and the large extraction is carried out, thus obtaining the homologous recombinant vector pHS-AVC-YW077cecropin (figure 1, SEQ ID NO: 4).
EXAMPLE 2 preparation of transgenic cell line specifically expressing Cecropin as antibacterial peptide
A50-gestational-age large white pig fetal fibroblast cell line with female gender is selected, and cell recovery, culture and passage are referred to 'finely compiled cell biology experimental guidelines' (J.S. Bonifis et al, translation of Octopus et al, 2007, science publishers). After the cells had grown and reached about 80%, the cells were collected by digestion (about 1X 10)6Separately), 2. mu.g of the vector pHS-AVC-YW077cecropin constructed in example 1 and 100. mu.L of a Nucleofector reagent were added and mixed, and the mixture was added to a cuvette and subjected to electroporation using the A-024 program. Then inoculating the mixture into a 10cm culture dish according to the ratio of 1:20, and carrying out inoculation at 37.5 ℃ and 5% CO2Culturing in an incubator. After 24h, the complete medium (10% FBS + DMEM) containing 500. mu.g/mL G418 was replaced, the medium was replaced every 3-4 days, and the G418 concentration increased to 800. mu.g/mL after 72 h. Colony point formation was visible by day 8 of culture. The colony sites were marked under the mirror, digested with cell cloning rings, and seeded into 24-well plates. When the confluency reaches 90%, the cells are digested, one part is left in the original well for continuous culture for identifying the integration site, and the other part is inoculated to a 6-well plate for cell cryopreservation.
And (3) monoclonal identification of positive cells:
the 414 cell clones obtained were identified. Short-arm primer SF: 5'-TGGGTGCTTGAGGTGGTCT-3', short-arm primer SR: 5'-CAAGCATAAAAACGCACCAATTGTC-3', PCR the amplification product size was 1300bp (FIG. 2, A). Long arm primer LF: 5'-GGAAGTGAGCAGGTAGAGGGTC-3', long-arm primer LR: 5'-ATCCAGTGTTGCCGTGAGC-3', PCR the amplification product size was 7975bp (FIG. 2, B). Digesting the monoclonal cell in 12-pore plate, extracting genome, using wild pig fibroblast genome as negative control, amplifying long and short arms with KOD polymerase, and detecting with 0.8% agarose electrophoresis. The results are shown in FIG. 2. Wherein the number of positive clones is 8, the state is good, and the method is suitable for nuclear transfer donor cells. Namely, the efficiency of site-directed integration of the Cecropin gene of the present invention is about 1.9%.
The results show that the mammary gland specificity expression antibacterial peptide Cecropin transgenic pig cell line is successfully constructed and obtained.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference documents:
[1]Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001.
sequence listing
<110> university of agriculture in China
<120> mammary gland specificity expression antibacterial peptide Cecropin gene homologous recombinant vector and application
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ttgctcccgc agagggagga gcgcttcctg ctcttctctt gtcactgatt ggccgcttct 180
cctcccgccg tgtgtgaaaa cacaaatggc gtgttttggt tggagtaaag ctcctgtcag 240
ttacagcctc gggagtgcgc agcctcccag gaactctcgc attgccccct gggtgggtag 300
gtaggtgggg tggagagagc tgcacaggcg ggcgctgtcg gcctcctgcg gggggagggg 360
agggtcagtg aaagtggctc ccgcgcgggc gtcctgccac cctcccctccgggggagtcg 420
gtttacccgc cgcctgctcg gctttggtat ctgattggct gctgaagtcc tgggaacggc 480
cccttgttat tggcttgggt cccaaatgag cgaaaaccac tacgcgagtc ggcagggagg 540
cggtctttgg tacggccctc cccgaggcca gcgccgcagt gtctggcccc tcacccctgc 600
gcaacgtggc aggaagcgcg cgcaggaggc gggggcgggc tgccgggccg aggcttctgg 660
gtggtggtga ctgcggctcc gccctgggcg tccgccgcct gaaggacgag actagctctc 720
tacctgctct cggacccgtg ggggtggggg gtggaggaag gagtgggggg tcggtcctgc 780
tggcttgtgg gtgggaggcg catgttctcc aaaaacccgc gcgagctgca atcctgaggg 840
agctgcagtg gaggaggcgg agagaaggcc gcacccttct ccgcaggggg aggggagtgc 900
cgcaatacct ttatgggagt tctctgctgc ctccttttcc taaggaccgc cctgggccta 960
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gaaaataact gacacaactc cattactggc tcctaactac ttaaacaatg cattctatca 180
tcacaaatgt gaaaaaggag ttccctcagt ggactaacct tatcttttct caacaccttt 240
ttctttgcac aattttccac acatgcctac aaaaagtact tctctgctca agtcacactg 300
agttgattgc tatttaccga aatcaaagta acattatcag atctctgtag ggtggttccc 360
tctggaatgc taccctccat agtccttacc cttcaagtaa agagcatgaa gactgaaata 420
tctcctctgt gatctgtcat cctttaagcc agaatccccc ataaaaaagt tagtattgct 480
ttctcctgat cccatagcag gttgaatcat agcacttatc aggttgttgt cattgcttgc 540
ttaaattctc ctaactattt ggagcttctt gagggcacag gttcttgttg agtcttgtac 600
ctaagcacct agtatagtcc ttgatgtcta gccaacccta aataaaatgc agtgagtgac 660
atgtagatgt ctttataagg tttgataggt tggtctctca aacagttctt ttgtatgttt 720
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gataattttc ttttcatact aagcagtaag gaaagaaaag ttgaaactct gtggtccatt 1020
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ctgagagcta aagccaacaa accagtactg tagataacct gctttggaca acaatgtgtt 1140
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cccctgaatt tcagtcctac aaagttttca gttttggaaa aactttcatc agagagggca 1320
ctaagttaca ggaagccatc acaaagtaag ttttcatctg atgaattata aatttaagat 1380
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cagtgcctta acacagacct gggttctcag taaatgttga ttttattgat ttatatgtga 2160
aattgttttt caaataatag tttttaagtc catagaaaca atgcttcttt tatggagata 2220
ctttaggatc atacttgtaa cccaagttgc ctaatactct gttcataaag aaaactcatg 2280
cctcatggtc tctgaataat acatctgtct accattgagc tcttccttgg gtttcctgtg 2340
caaaccattg cacttatcct cttcctgtgc tatacttcct caggctttat tacagttttt 2400
aaaataaacc aactgtctat ctctctttga agtagagcca taataattgc atgagaacac 2460
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tgtggcgcag tgggttaagg atccagcgtt gccatgagct gtggtatagg tccatatgtg 2580
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agggtaagac tagtttgttt taggccagac ttaattgtac agcccattgt ccagccacat 2880
actcaggagt ctcatacttt gcaggctaaa aattcttgat tttgttacct agtagtgtac 2940
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tttattttca tttatttatt tatttatttt tgctttttta gggccacact tgtggcatat 3060
ggaaattcct gggctagggg tcaaatcagg gcttcagctg ctggcctatg ccacaacaac 3120
acgggatcag agctgcatct gcaatctata ccacagcttt tggcaacccc gtatccttaa 3180
cccaatgaat actagttggg ttcttaaccc gctaagccat aattggaact cccatgatcc 3240
ccattcttga tgaagtgggt gagaaactgg tttattaaag aactattaat agcatggaat 3300
gaaatattta ggtctgtttt atctaggcca gagtcaaact tgatgactta ttactggtgt 3360
catggtgtat acctttcttg ctgactagag actatttctg cctctctgat tgtatctcaa3420
agtcatggtt gcatttaatc ctacttttcc agccttttcc cccagatatt tagtttgtac 3480
tggtacttag tcaccctttg ttttctaggt ttgtttgttt tttttttttt aaacatgttt 3540
gtgattggct tttcctgctg acaacagtaa cacattttct cagcacattt atagaattcg 3600
ttattttact agctaactaa ccccaccctc tagtgtaggg acagtggttt tttcttttta 3660
ttaacctttg gttaaaatcc caccccagac tttttattac aagtaatagc aatttaccct 3720
agtgcatacc ataggcaaaa gctaatggaa atgtaggtta gaagaaatga actaatattc 3780
gttgagtgac agttctgtac cagttagtat ggcagacttg ccagtgagtt tagtgtggtt 3840
ctgatgtata aaacttcatc ttacagagtg tgaaactaaa gctcagtgag gtttaataat 3900
tctcctgtaa acacagcaat caggagctac agattcaaaa ttctaatctg acttacctag 3960
tcggtatccc ttacttagct tagggtacca tgtttctcta tagaaacagt cttttaggaa 4020
atttaatctg gctacagata caggatgggt tgtagggttg aaggcagaga gaccaaactt 4080
aggagattct tttacagtag tcctgacagg aagcgatggg gtctgttata caatgatggc 4140
aatcaaaatg gaaaggaagt taaaggactc tgaacaccag tcactgaaaa gaatggtctc 4200
aacacatcac ttttcaagga agccttcctt gatgccctta actccacgat gttggattcc 4260
ccacttgaaa gctgtcatag cacttctcac aactgtatta gattagtaag tgggggaatt 4320
ttttttttgc tttttaaggt tgctccttga ggggatgatg gtgcttatat tcctgttgtc 4380
taatcatagg gaggtcttca ggtcaaaact attttcatag gaattctctg gtggcctatg 4440
ggttaaggat ttggcattgt cactgctgtg ggtgtggcca gagcccattt ttttttctca 4500
caggtgtgta ggggctatgt gaattttcca gaatttccca aggttgtagg tttatgtttt 4560
gtaactttag aaattaactt tctttaggtt atacctctaa aatcccacca accttcagaa 4620
ttatgtaatt gctactacaa aactctgtag ttaaatacat tctgtggtct tgttatgaaa 4680
taaagtttta aatattttct cacaacttat taacttttaa atttaattac aacttgtttt 4740
agaattttgg ggggggggct tcctcaaggt atatggtgtt ctgggccagg gatcagatgt 4800
gagccactgt ttgacctaca ctgcagctat ggcaacgctg gatcctttaa tcccttgtgt 4860
ggtgccaggg actgaacctg cgttctggca ctgcagagag agatgctgct gatcttactc 4920
agccacagca ggaaccccta ggatttaata atttattcaa aataaaccaa tttttcttat 4980
tatataaata ctatatatag atactataag taatattttt taagaataga tcattggctt 5040
aaaaaatgac atgttcagcc cacaactaca ccatctatgt ttaaaaatgc taagaagatg 5100
aagttaatag aaactataag gaaaacctac tgaagtatct taaatgataa attatataac 5160
atatggctgg cagatcatac atattagtgg agaaacttta caaaaagcca taaacagctg 5220
atgctcttga atgatgaaaa gtaaaaattc atggtagtgc tgctttataa caatatagta 5280
acttgttaag atttatgctc agacatgaag tctgttaata ccacattggt agcactgtat 5340
gtgtgcctca caatagatga gttttcaaac atggttgggc ttgttatgtt gcttttacct 5400
gtctggcatt agcagctagt cagactctta acatgaagtg cgacaaacac aacatgtcct 5460
attaaataac tttaaaatct taacggttta tcctgacctt ttcactaatg gtgaaacgca 5520
aacgtgggta aaactgccag gacttggcag aaagaaagac agtggcacaa aactagcagt 5580
cattttctgc catccatttg cagaataggg ggtggcagtt tcacttaaag aatgtccttg 5640
tgtgggagtt cccgtcgtgg ttcagtggtt aatgaatccg actaggtacc gtgaggttgc 5700
aggtttgatc cctggccttg ctcagtgggt taaggatccc gagttgctgt ggctctggcg 5760
taggccggtg gctacggctc caattcagcc cctagcctgg gaacctccat attgctgagg 5820
gagtggccca agaaatggca aaaggaaaaa aaaaaggtcc ttgtgaaaac aaattatttg 5880
attaaatctg gaccgtgagt acataataca catatgggtg atgaaatgga aagtttgtgg 5940
tacttcatat gctaaaaagt ataatggttg tctcaggaaa aaacattcaa gacagttgag 6000
ttgcaagctg atttagctcc tattttcttg gaatactaat tttacttgaa agattgactg 6060
tcaaattttg attgtcacag ttggatattt ggcagacatt ttttcaaata cgaaggaagt 6120
tagcctttca aggaaaactg acagcagttg ttgtcaatga taaaattcaa acttggaagt 6180
gaaaatacag aattctgggg aaacatctat tagcaagagg ctgactatca gtactgtccc 6240
tagtg 6245
<210>3
<211>213
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
atgttcctca tatatctgtt cgtgcaaacg gcagaaagca gctggctcag caaaactgcc 60
aagaaacttg agaactcagc aaagaaacgc atatctgaag gaattgcaat cgctatacaa 120
ggcggtccgc gccgacgtcg tttcgtcgca gagcaggacg cgattcactc tcgtgttagc 180
cgagaagtgc ctaccctgtc cgactcagtt tga 213
<210>4
<211>33204
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
gtatcactag tattacagag gtaagttata acagtcgcct aacccaattg catcgatggt 60
accagtgaaa gtctgcaatg aattcgagtt ggcgcgcctg tcattctaaa tctctctttc 120
agcctaaagc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa 180
gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct 240
gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc 300
tgccccctag cgggggaggg acgtaattac atccctgggg gctttgggag ggggctgtcc 360
ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct ttcagcctaa agctttttcc 420
ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg 480
atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg 540
gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga 600
gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag ctcccaggcg 660
cgccaactcg gtaccgtatc gatgtcgacg cggccgcact gaccctatag tgagtcgtat 720
taatttaaat cataccaaca tggtcaaata aaacgaaagg ctcagtcgaa agactgggcc 780
tttcgtttta atctgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat 840
cactaccggg cgtatttttt gagttatcga gattttcagg agctaaggaa gctaaaatgg 900
agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt 960
ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta 1020
cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca 1080
ttcttgcccg cctgatgaat gctcatccgg aatttcgtat ggcaatgaaa gacggtgagc 1140
tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt 1200
tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc 1260
aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata 1320
tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca 1380
atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca 1440
aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg 1500
gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaaccta 1560
ggtgacagaa gtcaaaagcc tccggtcgga ggcttttgac tttctgctag atcggccgca 1620
tcgaatataa cttcgtataa tgtatgctat acgaagttat tagcgatgag ctcggacttc 1680
cattgttcat tccacggaca aaaacagaga aaggaaacga cagaggccaa aaagctcgct 1740
ttcagcacct gtcgtttcct ttcttttcag agggtatttt aaataaaaac attaagttat 1800
gacgaagaag aacggaaacg ccttaaaccg gaaaattttc ataaatagcg aaaacccgcg 1860
aggtcgccgc cccgtaacct gtcggatcac cggaaaggac ccgtaaagtg ataatgatta 1920
tcatctacat atcacaacgt gcgtggaggc catcaaacca cgtcaaataa tcaattatga 1980
cgcaggtatc gtattaattg atctgcatca acttaacgta aaagcaactt cagacaatac 2040
aaatcagcga cactgaatac ggggcaacct catgtccgag ctcgcgagct cgtcgacagc 2100
gacacacttg catcggatgc agcccggtta acgtgccggc acggcctggg taaccaggta 2160
ttttgtccac ataaccgtgc gcaaaatgtt gtggataagc aggacacagc agcaatccac 2220
agcaggcata caaccgcaca ccgaggttac tccgttctac aggttacgac gaccatgtca 2280
atacttgccc ttgacaggca ttgatggaat cgtagtctca cgctgatagt ctgatcgaca 2340
atacaagtgg gaccgtggtc ccagaccgat aatcagaccg acaacacgag tgggatcgtg 2400
gtcccagact aataatcaga ccgacgatac gagtgggacc gtggtcccag actaataatc 2460
agaccgacga tacgagtggg accgtggttc cagactaata atcagaccga cgatacgagt 2520
gggaccgtgg tcccagacta ataatcagac cgacgatacg agtgggacca tggtcccaga 2580
ctaataatca gaccgacgat acgagtggga ccgtggtccc agtctgatta tcagaccgac 2640
gatacgagtg ggaccgtggt cccagactaa taatcagacc gacgatacga gtgggaccgt 2700
ggtcccagac taataatcag accgacgata cgagtgggac cgtggtccca gtctgattat 2760
cagaccgacg atacaagtgg aacagtgggc ccagagagaa tattcaggcc agttatgctt 2820
tctggcctgt aacaaaggac attaagtaaa gacagataaa cgtagactaa aacgtggtcg 2880
catcagggtg ctggcttttc aagttcctta agaatggcct caattttctc tatacactca 2940
gttggaacac gagacctgtc caggttaagc accattttat cgcccttata caatactgtc 3000
gctccaggag caaactgatg tcgtgagctt aaactagttc ttgatgcaga tgacgtttta 3060
agcacagaag ttaaaagagt gataacttct tcagcttcaa atatcacccc agcttttttc 3120
tgctcatgaa ggttagatgc ctgctgctta agtaattcct ctttatctgt aaaggctttt 3180
tgaagtgcat cacctgaccg ggcagatagt tcaccggggt gagaaaaaag agcaacaact 3240
gatttaggca atttggcggt gttgatacag cgggtaataa tcttacgtga aatattttcc 3300
gcatcagcca gcgcagaaat atttccagca aattcattct gcaatcggct tgcataacgc 3360
tgaccacgtt cataagcact tgttgggcga taatcgttac ccaatctgga taatgcagcc 3420
atctgctcat catccagctc gccaaccaga acacgataat cactttcggt aagtgcagca 3480
gctttacgac ggcgactccc atcggcaatt tctatgacac cagatactct tcgaccgaac 3540
gccggtgtct gttgaccagt cagtagaaaa gaagggatga gatcatccag tgcgtcctca 3600
gtaagcagct cctggtcacg ttcattacct gaccataccc gagaggtctt ctcaacacta 3660
tcaccccgga gcacttcaag agtaaacttc acatcccgac cacatacagg caaagtaatg 3720
gcattaccgc gagccattac tcctacgcgc gcaattaacg aatccaccat cggggcagct 3780
ggtgtcgata acgaagtatc ttcaaccggt tgagtattga gcgtatgttt tggaataaca 3840
ggcgcacgct tcattatcta atctcccagc gtggtttaat cagacgatcg aaaatttcat 3900
tgcagacagg ttcccaaata gaaagagcat ttctccaggc accagttgaa gagcgttgat 3960
caatggcctg ttcaaaaaca gttctcatcc ggatctgacc tttaccaact tcatccgttt 4020
cacgtacaac attttttaga accatgcttc cccaggcatc ccgaatttgc tcctccatcc 4080
acggggactg agagccatta ctattgctgt atttggtaag caaaatacgt acatcaggct 4140
cgaacccttt aagatcaacg ttcttgagca gatcacgaag catatcgaaa aactgcagtg 4200
cggaggtgta gtcaaacaac tcagcaggcg tgggaacaat cagcacatca gcagcacata 4260
cgacattaat cgtgccgata cccaggttag gcgcgctgtc aataactatg acatcatagt 4320
catgagcaac agtttcaatg gccagtcgga gcatcaggtg tggatcggtg ggcagtttac 4380
cttcatcaaa tttgcccatt aactcagttt caatacggtg cagagccaga caggaaggaa 4440
taatgtcaag ccccggccag caagtgggct ttattgcata agtgacatcg tccttttccc 4500
caagatagaa aggcaggaga gtgtcttctg catgaatatg aagatctggt acccatccgt 4560
gatacattga ggctgttccc tgggggtcgt taccttccac gagcaaaaca cgtagcccct 4620
tcagagccag atcctgagca agatgaacag aaactgaggt tttgtaaacg cctcctttat 4680
gggcagcaac cccgatcacc ggtggaaata cgtcttcagc acgtcgcaat cgcgtaccaa 4740
acacatcacg catatgatta atttgttcaa ttgtataacc aacacgttgc tcaacccgtc 4800
ctcgaatttc catatccggg tgcggtagtc gccctgcttt ctcggcatct ctgatagcct 4860
gagaagaaac cccaactaaa tccgctgctt cacctattct ccagcgccgg gttattttcc 4920
tcgcttccgg gctgtcatca ttaaactgtg caatggcgat agccttcgtc atttcatgac 4980
cagcgtttat gcactggtta agtgtttcca tgagtttcat tctgaacatc ctttaatcat 5040
tgctttgcgt ttttttatta aatcttgcaa tttactgcaa agcaacaaca aaatcgcaaa 5100
gtcatcaaaa aaccgcaaag ttgtttaaaa taagagcaac actacaaaag gagataagaa 5160
gagcacatac ctcagtcact tattatcact agcgctcgcc gcagccgtgt aaccgagcat 5220
agcgagcgaa ctggcgagga agcaaagaag aactgttctg tcagatagct cttacgctca 5280
gcgcaagaag aaatatccac cgtgggaaaa actccaggta gaggtacaca cgcggatagc 5340
caattcagag taataaactg tgataatcaa ccctcatcaa tgatgacgaa ctaacccccg 5400
atatcaggtc acatgacgaa gggaaagaga aggaaatcaa ctgtgacaaa ctgccctcaa 5460
atttggcttc cttaaaaatt acagttcaaa aagtatgaga aaatccatgc aggctgaagg 5520
aaacagcaaa actgtgacaa attaccctca gtaggtcaga acaaatgtga cgaaccaccc 5580
tcaaatctgt gacagataac cctcagacta tcctgtcgtc atggaagtga tatcgcggaa 5640
ggaaaatacg atatgagtcg tctggcggcc tttctttttc tcaatgtatg agaggcgcat 5700
tggagttctg ctgttgatct cattaacaca gacctgcagg aagcggcggc ggaagtcagg 5760
catacgctgg taactttgag gcagctggta acgctctatg atccagtcga ttttcagaga 5820
gacgatgcct gagccatccg gcttacgata ctgacacagg gattcgtata aacgcatggc 5880
atacggattg gtgatttctt ttgtttcact aagccgaaac tgcgtaaacc ggttctgtaa 5940
cccgataaag aagggaatga gatatgggtt gatatgtaca ctgtaaagcc ctctggatgg 6000
actgtgcgca cgtttgataa accaaggaaa agattcatag cctttttcat cgccggcatc 6060
ctcttcaggg cgataaaaaa ccacttcctt ccccgcgaaa ctcttcaatg cctgccgtat 6120
atccttactg gcttccgcag aggtcaatcc gaatatttca gcatatttag caacatggat 6180
ctcgcagata ccgtcatgtt cctgtagggt gccatcagat tttctgatct ggtcaacgaa 6240
cagatacagc atacgttttt gatcccggga gagactatat gccgcctcag tgaggtcgtt 6300
tgactggacg attcgcgggc tatttttacg tttcttgtga ttgataaccg ctgtttccgc 6360
catgacagat ccatgtgaag tgtgacaagt ttttagattg tcacactaaa taaaaaagag 6420
tcaataagca gggataactt tgtgaaaaaa cagcttcttc tgagggcaat ttgtcacagg 6480
gttaagggca atttgtcaca gacaggactg tcatttgagg gtgatttgtc acactgaaag 6540
ggcaatttgt cacaacacct tctctagaac cagcatggat aaaggcctac aaggcgctct 6600
aaaaaagaag atctaaaaac tataaaaaaa ataattataa aaatatcccc gtggataagt 6660
ggataacccc aagggaagtt ttttcaggca tcgtgtgtaa gcagaatata taagtgctgt 6720
tccctggtgc ttcctcgctc actcgaccgg gagggttcga gaaggggggg cacccccctt 6780
cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat 6840
tggtttaaaa gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt 6900
gcaaatgctg gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca 6960
tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc 7020
gcccctcaag tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg 7080
gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc 7140
cggccgaaat cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca 7200
agtgtcaacg tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca 7260
acgccggcgg ccggccgcgg tgtctcgcac acggcttcga cggcgtttct ggcgcgtttg 7320
cagggccata gacggccgcc agcccagcgg cgagggcaac cagccgaggg cttcgccctg 7380
tcgctcgact gcggcgagca ctactggctg taaaaggaca gaccacatca tggttctgtg 7440
ttcattaggt tgttctgtcc attgctgaca taatccgctc cacttcaacg taacaccgca 7500
cgaagatttc tattgttcct gaaggcatat tcaaatcgtt ttcgttaccg cttgcaggca 7560
tcatgacaga acactacttc ctataaacgc tacacaggct cctgagatta ataatgcgga 7620
tctctacgat aatgggagat tttcccgact gtttcgttcg cttctcagtg gataacagcc 7680
agcttctctg tttaacagac aaaaacagca tatccactca gttccacatt tccatataaa 7740
ggccaaggca tttattctca ggataattgt ttcagcatcg caaccgcatc agactccggc 7800
atcgcaaact gcacccggtg ccgggcagcc acatccagcg caaaaacctt cgtgtagact 7860
tccgttgaac tgatggactt atgtcccatc aggctttgca gaactttcag cggtataccg 7920
gcatacagca tgtgcatcgc ataggaatgg cggaacgtat gtggtgtgac cggaacagag 7980
aacgtcacac cgtcagcagc agcggcggca accgcctccc caatccaggt cctgaccgtt 8040
ctgtccgtca cttcccagat ccgcgctttc tctgtccttc ctgtgcgacg gttacgccgc 8100
tccatgagct tatcgggtcc actcgcgaac gcagaaaggc ccacccgaag gtgagccagt 8160
gtgattacat ttgcggccta actgtggcca gtccagttac gctggagtca ctagtattta 8220
ggtgacacta tagaagcggc cttggcgcgc cgctagaggt ttaccgagct cttattggtt 8280
ttcaaacttc attgactgtg ccaagcttct gacctcttct cttcctccca cagggcctcg 8340
tttgttacgc tggaagggga agaggtggtg agaggcaggc gggagtgcgg cccgccctgc 8400
ggcaaccgga gggggaggga gaagggagcg gaaaagcctg gaatacggac ggagccattg 8460
ctcccgcaga gggaggagcg cttcctgctc ttctcttgtc actgattggc cgcttctcct 8520
cccgccgtgt gtgaaaacac aaatggcgtg ttttggttgg agtaaagctc ctgtcagtta 8580
cagcctcggg agtgcgcagc ctcccaggaa ctctcgcatt gccccctggg tgggtaggta 8640
ggtggggtgg agagagctgc acaggcgggc gctgtcggcc tcctgcgggg ggaggggagg 8700
gtcagtgaaa gtggctcccg cgcgggcgtc ctgccaccct cccctccggg ggagtcggtt 8760
tacccgccgc ctgctcggct ttggtatctg attggctgct gaagtcctgg gaacggcccc 8820
ttgttattgg cttgggtccc aaatgagcga aaaccactac gcgagtcggc agggaggcgg 8880
tctttggtac ggccctcccc gaggccagcg ccgcagtgtc tggcccctca cccctgcgca 8940
acgtggcagg aagcgcgcgc aggaggcggg ggcgggctgc cgggccgagg cttctgggtg 9000
gtggtgactg cggctccgcc ctgggcgtcc gccgcctgaa ggacgagact agctctctac 9060
ctgctctcgg acccgtgggg gtggggggtg gaggaaggag tggggggtcg gtcctgctgg 9120
cttgtgggtg ggaggcgcat gttctccaaa aacccgcgcg agctgcaatc ctgagggagc 9180
tgcagtggag gaggcggaga gaaggccgca cccttctccg cagggggagg ggagtgccgc 9240
aataccttta tgggagttct ctgctgcctc cttttcctaa ggaccgccct gggcctagaa 9300
aaatccctcc ctcccccgcg atctcgtcat cgcctccatg tcagtttgct ccttctcgat 9360
tatgggcggg attcttttgc cctggcttaa cctgattctt gggcgttgtc ctgcagggga 9420
ttgagcaggt gtacgaggac gagcccaatt tctctatatt cccacagtct tgagtttgtg 9480
tcacaaaata attatagtgg ggtggagatg ggaaatgagt ccaggcaaca cctaagctac 9540
cgttcgtata gcatacatta tacgaagtta tgacaattgg tgcgttttta tgcttgtagt 9600
attgtataat gtttttaaga tccaagcttc tgacctcttc tcttcctccc acagggcctc 9660
gagacaattg catcgatggt accagtgaaa gtctgcaatg aattcgagtt ggcgcgcctg 9720
tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca ggtgtctgca 9780
ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc 9840
ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg 9900
agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg 9960
gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct 10020
ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga 10080
gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac 10140
gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc 10200
tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg 10260
tccccgtgag ctcccaggcg cgccaactcg gtaccgtatc gatgtcgacg ttaacgctag 10320
tgatctagag ctaggcgcgc ctccgccagc acaggaatgg gggaggggtg ggtgacgagg 10380
atgaacaccg gagtccctgg aggaagggaa gcagggtatc tccatctgag gctctgtctt 10440
tgaggagagg tggagagctg gggaagtctt gtgtgagggg attggggctc aggagggggt 10500
tggggagcag gaagttgtcc ccaggggagc catcctggcc cattcaaggg ttgagtactt 10560
gtttagggtt agagctgccc cctctgggga ccaggattgt ccagccaagg ccattgtcct 10620
gcccccttcc cccagtccct cccaggcccc tttgaacctg aagtcagata tttcttctct 10680
ctacccacct cccacccgtt gggtttctcc acccaggaac taggctggaa gcctgggatg 10740
aggaggtggg gggagggaga actgagaatc ttgaggaaag aggccccggc cttaactgtg 10800
aggggatgga gcctgggtgc aggtcttatg ggggttgggg ggtggttagt gtctaatcta 10860
ccaacctgga caacacaaga tggaatactg tgctctgaaa acgcagagcc agcacttctc 10920
tggggtctct ggggacatat ctggttgggg ctcggggtcc catggtgtag agcctctaaa 10980
ctctggagga ctggaggtgc aatggctgtc ttgtcctggc cttggacatg ggctgaaata 11040
ctgggttcac ccatatctag gactctagac gggtgggtaa gcaagaactg aggagtggcc 11100
ccagaaataa ttggcacacg aacattcaat ggatgtttta ggctctccag aggatggctg 11160
agtgggctgt aaggacaggc cgagagggtg cagtgccaac aggctttgtg gtgcgatggg 11220
gcatccgagc aactggtttg tgaggtgtcc ggtgacccaa ggcaggggtg agaggacctt 11280
gaaggttgaa aatgaaggcc tcctggggtc ccgtcctaag ggttgtcctg tccagacgtc 11340
cccaacctcc gtctggaaga cacaggcaga tagcgctcgc ctcagtttct cccaccccca 11400
cagctctgct cctccaccca cccagggggc ggggccagag gtcaaggcta gagggtggga 11460
ttggggaggg agaggtgaaa ccgtccctag gtgagccgtc tttccaccag gcccccggct 11520
cggggtgccc accttccccg gccggccatg cccaagaaga agaggaaggt gtccaattta 11580
ctgaccgtac accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggttcgc 11640
aagaacctga tggacatgtt cagggatcgc caggcgtttt ctgagcatac ctggaaaatg 11700
cttctgtccg tttgccggtc gtgggcggca tggtgcaagt tgaataaccg gaaatggttt 11760
cccgcagaac ctgaagatgt tcgcgattat cttctatatcttcaggcgcg cggtctggca 11820
gtaaaaacta tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg 11880
ccacgaccaa gtgacagcaa tgctgtttca ctggttatgc ggcggatccg aaaagaaaac 11940
gttgatgccg gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga tttcgaccag 12000
gttcgttcac tcatggaaaa tagcgatcgc tgccaggata tacgtaatct ggcatttctg 12060
gggattgctt ataacaccct gttacgtata gccgaaattg ccaggatcag ggttaaagat 12120
atctcacgta ctgacggtgg gagaatgtta atccatattg gcagaacgaa aacgctggtt 12180
agcaccgcag gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg 12240
atttccgtct ctggtgtagc tgatgatccg aataactacc tgttttgccg ggtcagaaaa 12300
aatggtgttg ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct ggaagggatt 12360
tttgaagcaa ctcatcgatt gatttacggc gctaaggtaa atataaaatt tttaagtgta 12420
taatgtgtta aactactgat tctaattgtt tgtgtatttt aggatgactc tggtcagaga 12480
tacctggcct ggtctggaca cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct 12540
ggagtttcaa taccggagat catgcaagct ggtggctgga ccaatgtaaa tattgtcatg 12600
aactatatcc gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc 12660
gattagccat taacgcgtaa atgattgcta taattatttg atatttatgg tgacatatga 12720
gaaaggattt caacatcgac ggaaaatatg tagtgctgtc tgtaagcact aatattcagt 12780
cgccagccgt cattgtccac tgtaaagctg agcggcaata aaaagacaga ataaaacgca 12840
cgggtgttgg gtcgtttgtt ccctgcaggc gtcatctagc ttggctgcag gtcgtcgaaa 12900
ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 12960
ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc 13020
ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc 13080
ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa 13140
gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta 13200
ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg 13260
ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga 13320
aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc 13380
tcctcttcct catctccggg cctttcgacc tgcagcctgt tgacaattaa tcatcggcat 13440
agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggga tcggccattg 13500
aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 13560
actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 13620
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 13680
aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 13740
ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 13800
tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 13860
tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 13920
gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 13980
aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg 14040
atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 14100
tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 14160
tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 14220
tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 14280
tcttctgatg aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg 14340
gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg 14400
gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt 14460
gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat 14520
catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg 14580
gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct 14640
gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt 14700
tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga 14760
tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc 14820
ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 14880
ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 14940
tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 15000
gggaaacctg tcgtgccagc ggatcgacag tactaagctt ggtctaatac ccaatctctc 15060
gtcttatcca gatgttttat acgccaagct tctgacctct tctcttcctc ccacagggcc 15120
tcgagacaat tgcatcgatg gtaccagtga aagtctgcaa tgaattcgag ttggcgcgcc 15180
tgtcattcta aatctctctt tcagcctaaa gctttttccc cgtatccccc caggtgtctg 15240
caggctcaaa gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt 15300
gcccgggctg tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc 15360
ggagccccgg gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg 15420
gggctttggg agggggctgt ccccgtgagc tcccaggcgc gcctgtcatt ctaaatctct 15480
ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc 15540
gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc 15600
acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc 15660
gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc 15720
tgtccccgtg agctcccagg cgcgccaact cggtaccgta tcgatgtcga cgttaacgct 15780
agtgatatca actttgtata gaaaagttgg ctccgaattt cataacttcg tataatgtat 15840
gctatacgaa gttatgtgca catgaacaca tgatgtggag ctcaggatag aggaggagga 15900
ttttgttaat ccttcttcca acaagagttc caagccatgg ctgggtgcaa tggtttatgc 15960
ctgtaattcc agcactttgg gaggccaagg tgggaggatc gcttgggccc aggaggtcaa 16020
ggctgcagtg agctgtgatt gcaccaccac tgcataccag cctaggtgac agagggagac 16080
cctgtcggta aaaaaaaaaa aaaaaaaaag aaaagaaaag aaaagaaaaa aagaattcct 16140
ggccagaaag ccagagggaa cttttgttgt ccctagtgct caggtaattg gtcactgggt 16200
tcatgctccc aagcctttac ttgtttggtt gtttacttta cagaatgctc tacctgtaga 16260
aatcccaccc ttccttcagg tacccaggct aaatcttcct ggagttcttc tggagccctc 16320
atgatgggtg ctggtcattc ctcctctggg ccctcacact cacttgcttt atgctgcttg 16380
gcctcttctt agggagcctc tgtgatagaa gtgcattcct gtgactgtcc ctcccccata 16440
gtgctgtgag ctcctgaggg caaggacaga cccctcttct ctgtctctca tggcatctac 16500
cagttctggc atgaatgaat gaatgaatga atgaatgaat gctgttaata gatcagtgaa 16560
acttcattgt tttttggggc agactcatcc cctacatcag cctcctctgc aaatggcctt 16620
gaggggctgc tctgcctgtg tccagatgct cacatccctg ccctggggct gggctgttcc 16680
aacaggcaca gcaggaaaac agtctgccct gttgcctacc catgctgcct tcagggcact 16740
ccttaagctg agcctcttgg tagtggcccc aggttctctg tgttcttgcc aacactgtaa 16800
catacttaag agggcccagg gccctctgac ctcccagtca tcctttttta aaatttggac 16860
tcagaaaaag agacacgggt tatgatgttt cttaattctt ttataatgat gaaaaggcaa 16920
agtcttgttg ccaatttagg tacaaagatg cttcagcact cctgggaatt agtcaatttt 16980
gtatttcttc agtatttttg aaagaactta ttgcaattat tgatgatggc aactttaaat 17040
ggtgcaatat catgtttcca aacaatgaga gaccttggat ctgtcacccc aaaacccagc 17100
tggtgattct agcaccaaat ctcagacccc agtctcatgg caggcaagca tgatccctga 17160
ttagcctcca ccctgtgcct tggcaggatc atgcccagaa atggaggggc tccccagcct 17220
ccttaatggc ctctccacct tgggctgagc ctgcttctcc atggctaggt gcccactgca 17280
tgctcactct tggcagagct ggctccctgg ggcattgcct atctgctcca ggaaatgctt 17340
tttagtacca agtagtctaa gcagagtcaa gaccagcttt tcagaataag cagatttcag 17400
agtcacatat tggtcagact cacctgccaa gaaaggtgtt caggtgacct attatttccc 17460
tcactgttag tacctttttt cttcacttag ttaccctttt tttttttggt gggggttaca 17520
gagtcttact ctattgccca ggctgcagtg cagtgacatg atcatggctc actgccacct 17580
tcatctccca ggctcaaatg gtcctcccac tttagcctcc caagtagctg ggaccatagg 17640
catacaccac catgctgggc taatttttgt attttttgta gagatggggg tttccctatg 17700
ttgcccaggc tagtcttgaa ctcctgggct caagcgatcc tcccatcttg gcctcccaaa 17760
gtgctgggat tacaggcatg agccactgtg ccctgcctag ttactcttgg gctaagttca 17820
catccataca cacaggatat tctttctgag gcccccaatg tgtcccacag gcaccatgct 17880
gtatgtgaca ctcccctaga gatggatgtt tagtttgctt ccaactgatt aatggcatgc 17940
agtggtgcct ggaaacattt gtacctgggg tgctgtgtgt catgggaatg tatttacgag 18000
atgtattctt agaagcagta ttctagcttt tgaattttaa aatctgacat ttatggcgat 18060
tgttaaaatg aggttaccat ttcctactga atactatcaa caccaaaaaa gaagaaggag 18120
gagatggaga aaaaaaagac aaaaaaaaaa aaaagtggta gggcatctta gccatagggc 18180
atctttctca ttggcaaata agaacatgga accagccttg ggtggtggcc attcccctct 18240
gaggtccctg tctgttttct gggagctgta ttgtgggtct cagcagggca gggagatacc 18300
ccatgggcag cttgcctgag actctgggca gcctctcttt tctctgtcag ctgtccctag 18360
gctgctgctg ggggtggtcg ggtcatcttt tcaactctca gctcactgct gagccaaggt 18420
gaaagcaaat ccacctgccc taactggctc ctaggcacct tcaaggtcat ctgctgaaga 18480
agatagcagt ctcacaggtc aaggcgatct tcaagtaaag accctctgct ctgtgtcctg 18540
ccctctagaa ggcactgaga ccagagctgg gacagggctc agggggctgc gactcctagg 18600
ggcttgcaga ctagtgggag agaaagaaca tcgcagcagc caggcagaac caggacaggt 18660
gaggtgcagg ctggctttcc tctcgcagcg cggtgtggag tcctgtcctg cctcagggct 18720
tttcggagcc tggatcctca aggaacaagt agacctggcc gcggggagtg gggagggaag 18780
gggtgtctat tgggcaacag ggcggggcaa agccctgaat aaaggggcgc agggcaggcg 18840
caagtggcag agccttcgtt tgccaagtcg cctccagacc gcagacgtaa ctgatcataa 18900
ttcgacccaa gtttgtacaa aaaagcaggc tgattaccgg agaattcatg ttcctcatat 18960
atctgttcgt gcaaacggca gaaagcagct ggctcagcaa aactgccaag aaacttgaga 19020
actcagcaaa gaaacgcata tctgaaggaa ttgcaatcgc tatacaaggc ggtccgcgcc 19080
gacgtcgttt cgtcgcagag caggacgcga ttcactctcg tgttagccga gaagtgccta 19140
ccctgtccga ctcagtttga gctagcccgg gtaactgaaa atccaccgga tctagataac 19200
tgatctaccc agctttcttg tacaaagtgg taaaccgaag aagatggccc agctccccaa 19260
gaaagcctca gccattcact gcccccagct cttctcccca ggtgtgttgg ggccttggcc 19320
tcccctgctg aaggtgggga ttgcccatcc atctgcttac aattccctgc tgtcgtctta 19380
gcaagaagta aaatgagaaa ttttgttgat attctctcct tataaagtgt cactcatctt 19440
ttctagaatt ttatactgaa atcacatgcc tgacaaaata cctgtacagt tggaccttcc 19500
cttccaagtt tttcaggtcc agcccctcct ctttcttgca gtcttgggta tgatgcccaa 19560
gggtctggaa tttaaggcca ggccaagcac cggttttcct aaggggatct tgttggttat 19620
tcacatagct gtctcagtgc acgtgcatgt atgtgcctgg gagtgtgtgc gtgtccccaa 19680
ggcagggcag gaagaacaag cataggaatt ataactgaaa tgtaggttca gttgctcatc 19740
acttgcagag tctaattaac aagagctagg tttggtataa agtgactttt attccaaagc 19800
taccttaggg gaagaagtac aggcttcttt ccttaagggt aactgcttca cctttggagc 19860
agaaagcaga ggcttttaaa tggggggcgt ggcatgcaag gcaggaagtg agcaggtaga 19920
gggtctgggt actggctttg gtgccttgtc taccaggcag ttgagttggt tactgctggt 19980
gcctttgtgg gcaaaactag gttgtaaaag cagccaaaaa atttccagat gggagagttt 20040
catagcgggc atattttggc ttgtgaattg actgttatgt ctcaaggtag tctcttggtg 20100
ggagagagtt ctgctctgta gcgtctaact aagagcacat agttagatga acttgccctg 20160
ttgggagtgt ctggtgaagg ggagataaaa gattataatt gcatttctaa agagctaagt 20220
aggaagcaga gaaaaggaga gagaagtgaa gacagaataa ttttaaataa tgaagagaga 20280
aatgaagaag gaataatttt cctgcttttc cattggttct ttggacataa caggcaaaag 20340
tgtcaatcag ggtgagactg ttccttcaag ggcagagatc agacaagtgt cctgaaactc 20400
tgcctccattgtgggggcaa ccatttggcc atgttcccca ctgtcagtga gatgaatagc 20460
tctgctgctg cctgcaaaag cctctgaaac ctgtcaggaa aggacattaa ttaaggtccc 20520
ataatgctag gatgaagctc ttttcctatt tgctactttg aattatggcc tgatatgttg 20580
tccatcctca ctaacatgag aagattagtc agtgagcaac atctgatggg gtagccacag 20640
cagtgataat gccagcactc ttaaacatga catttgaaca gtatgaactt tcttgtctaa 20700
ataagatact aaagtgaaat tgaggcccac ctatgaaatt ctcctgttcc aggttgaaaa 20760
gaaatggaac atcgctgtgt ctctctgttc acgctaggtt atattgctgt agcaaatggc 20820
ctccaaatca aaatcttagt ggctaacatg acaaaaatgt gtttctctct tgtgtccatc 20880
tccatcatgg gtcagcaatg gatctgtggc tcatgtcttt attctgagac ccaggctgaa 20940
ccagcagccc caattttggg catgttgggc ttgaggcaga ggggaaaata taagagacag 21000
aggcagctgc tctgcctatg gagtgctatt cttttattcc tttattttct taataaactg 21060
ctttaacttt aaaaaaagag acaaaagcat catacaataa tacttacagt gtctgcttgg 21120
cagaggccat atcatcttta tcatttcatt ggccagagaa ttcacatggc cacaacatca 21180
gtgaagtcgc ccaggaggaa ggggcagtga tagaggactg gggttagtgc aacagtgcaa 21240
ggaaaaatct tcagagcatt tcatagtgcc cagccagaag gccatcacag gaatgtccca 21300
cccacggtga attcccttat gtgagtgtaa aaggcaggcg agtttgtccc aagcttctga 21360
cctcttctct tcctcccaca gggcctcgag acgttagtaa ctgagctcag ttgccgtaat 21420
gctaatagct tccaaactag tgtctctgtc tccagtatct gataaatctt aggtgttgct 21480
gggacagttg tcctaaaatt aagataaagc atgaaaataa ctgacacaac tccattactg 21540
gctcctaact acttaaacaa tgcattctat catcacaaat gtgaaaaagg agttccctca 21600
gtggactaac cttatctttt ctcaacacct ttttctttgc acaattttcc acacatgcct 21660
acaaaaagta cttctctgct caagtcacac tgagttgatt gctatttacc gaaatcaaag 21720
taacattatc agatctctgt agggtggttc cctctggaat gctaccctcc atagtcctta 21780
cccttcaagt aaagagcatg aagactgaaa tatctcctct gtgatctgtc atcctttaag 21840
ccagaatccc ccataaaaaa gttagtattg ctttctcctg atcccatagc aggttgaatc 21900
atagcactta tcaggttgtt gtcattgctt gcttaaattc tcctaactat ttggagcttc 21960
ttgagggcac aggttcttgt tgagtcttgt acctaagcac ctagtatagt ccttgatgtc 22020
tagccaaccc taaataaaat gcagtgagtg acatgtagat gtctttataa ggtttgatag 22080
gttggtctct caaacagttc ttttgtatgt ttggtagtgc tctagattag cactggccag 22140
tataactctg atgatggaaa tgttctatag ctatgctgtc taatatggta gtcactacta 22200
acatatgtta ctgttgagcc ttggaaatat ggcttttgtg acaaaactga atttttcatg 22260
ctatgtaatt taagtctaaa ttgctactgt gtacattgtg gctgtagcca caaatttgtg 22320
ctgtggattg cagaataatt aatatggaca ttgataattt tcttttcata ctaagcagta 22380
aggaaagaaa agttgaaact ctgtggtcca tttaggttat atgtgtattt gtacttgatt 22440
ggtttgtttg aatacctatt tctatacttt agctgagagc taaagccaac aaaccagtac 22500
tgtagataac ctgctttgga caacaatgtg ttgactagct ggatttcacc aaagaatgcc 22560
taataaattt taagaaaatg agatttcatt aaaccataat actgacataa gtttagggaa 22620
gaatcagact atatctggtg tttgtgaaac tacccctgaa tttcagtcct acaaagtttt 22680
cagttttgga aaaactttca tcagagaggg cactaagtta caggaagcca tcacaaagta 22740
agttttcatc tgatgaatta taaatttaag atatatttta ataccaaaat tctttatggt 22800
ttatgtgcta acttaaaatt tctccttaaa atatgagaac taagtacaca attgtacttg 22860
gctgtttaat gcggattccc agtccctcac acagagattc tcaattaaga ttggagagca 22920
ggggttacta gaattctttt tcaggttcct tatatgcttc tgatttggtg gcctagaaat 22980
cacaatgcta gtgcagccct catggggcta cagtatacgt atctgaaaca tgattacatc 23040
agggaaactg tatgtctaat ctactttgtc cctaaaggaa gcattttgaa aggcagaaag 23100
taatatgtga tagtttttga aacttgtagg tcacattgtt tttaaaaggg atccaagtaa 23160
gttttttttt cttttgaggg ctacacctgt ggcacatgga ggttcccagg ctaggggtta 23220
aatcagaact gcagctgcca gcctatgcca gagccacagc aatgccagat ctgagctgtg 23280
tctgcaactg tgtagctcac agcaacgctg gatccttaac ccaatgagca aggccagaga 23340
ttgaacctac aacctcgtgg ttcctagtta gatttgtttc cgctgtgcca cgatgggaac 23400
tccaagtaat ttttttttga gcaaggaagt tacctttttt gtctgttttc ccactaaatg 23460
cattcctcaa ggattcccag ttcttgattc ctcagtgcct taacacagac ctgggttctc 23520
agtaaatgtt gattttattg atttatatgt gaaattgttt ttcaaataat agtttttaag 23580
tccatagaaa caatgcttct tttatggaga tactttagga tcatacttgt aacccaagtt 23640
gcctaatact ctgttcataa agaaaactca tgcctcatgg tctctgaata atacatctgt 23700
ctaccattga gctcttcctt gggtttcctg tgcaaaccat tgcacttatc ctcttcctgt 23760
gctatacttc ctcaggcttt attacagttt ttaaaataaa ccaactgtct atctctcttt 23820
gaagtagagc cataataatt gcatgagaac actgaaggtt tttaggcttt aatttttttt 23880
tttttaagat attcaaaatt tggagttctt gttgtggcgc agtgggttaa ggatccagcg 23940
ttgccatgag ctgtggtata ggtccatatg tggctcggat cctgcattgc tgtggctgtg 24000
gcagcagcca caggtacgat tagaccccta gcctgggacc ctccatatgc tgtgggcgcg 24060
gtcctagaaa agaaaaaaaa aaaaagaaaa gaaagatata caaaatttga actacgcatt 24120
gtttctctta acagttgtta tgtatggagg aggtttgtta taattacagt ttacaactct 24180
taatccagaa tatgttaggg atccacattc ccagggtaag actagtttgt tttaggccag 24240
acttaattgt acagcccatt gtccagccac atactcagga gtctcatact ttgcaggcta 24300
aaaattcttg attttgttac ctagtagtgt actgttcatg ttggggaact tttttctcca 24360
gaaaagttta ttatccatta tcctgcctcc tttttatttt catttattta tttatttatt 24420
tttgcttttt tagggccaca cttgtggcat atggaaattc ctgggctagg ggtcaaatca 24480
gggcttcagc tgctggccta tgccacaaca acacgggatc agagctgcat ctgcaatcta 24540
taccacagct tttggcaacc ccgtatcctt aacccaatga atactagttg ggttcttaac 24600
ccgctaagcc ataattggaa ctcccatgat ccccattctt gatgaagtgg gtgagaaact 24660
ggtttattaa agaactatta atagcatgga atgaaatatt taggtctgtt ttatctaggc 24720
cagagtcaaa cttgatgact tattactggt gtcatggtgt atacctttct tgctgactag 24780
agactatttc tgcctctctg attgtatctc aaagtcatgg ttgcatttaa tcctactttt 24840
ccagcctttt cccccagata tttagtttgt actggtactt agtcaccctt tgttttctag 24900
gtttgtttgt tttttttttt ttaaacatgt ttgtgattgg cttttcctgc tgacaacagt 24960
aacacatttt ctcagcacat ttatagaatt cgttatttta ctagctaact aaccccaccc 25020
tctagtgtag ggacagtggt tttttctttt tattaacctt tggttaaaat cccaccccag 25080
actttttatt acaagtaata gcaatttacc ctagtgcata ccataggcaa aagctaatgg 25140
aaatgtaggt tagaagaaat gaactaatat tcgttgagtg acagttctgt accagttagt 25200
atggcagact tgccagtgag tttagtgtgg ttctgatgta taaaacttca tcttacagag 25260
tgtgaaacta aagctcagtg aggtttaata attctcctgt aaacacagca atcaggagct 25320
acagattcaa aattctaatc tgacttacct agtcggtatc ccttacttag cttagggtac 25380
catgtttctc tatagaaaca gtcttttagg aaatttaatc tggctacaga tacaggatgg 25440
gttgtagggt tgaaggcaga gagaccaaac ttaggagatt cttttacagt agtcctgaca 25500
ggaagcgatg gggtctgtta tacaatgatg gcaatcaaaa tggaaaggaa gttaaaggac 25560
tctgaacacc agtcactgaa aagaatggtc tcaacacatc acttttcaag gaagccttcc 25620
ttgatgccct taactccacg atgttggatt ccccacttga aagctgtcat agcacttctc 25680
acaactgtat tagattagta agtgggggaa tttttttttt gctttttaag gttgctcctt 25740
gaggggatga tggtgcttat attcctgttg tctaatcata gggaggtctt caggtcaaaa 25800
ctattttcat aggaattctc tggtggccta tgggttaagg atttggcatt gtcactgctg 25860
tgggtgtggc cagagcccat ttttttttct cacaggtgtg taggggctat gtgaattttc 25920
cagaatttcc caaggttgta ggtttatgtt ttgtaacttt agaaattaac tttctttagg 25980
ttatacctct aaaatcccac caaccttcag aattatgtaa ttgctactac aaaactctgt 26040
agttaaatac attctgtggt cttgttatga aataaagttt taaatatttt ctcacaactt 26100
attaactttt aaatttaatt acaacttgtt ttagaatttt gggggggggg cttcctcaag 26160
gtatatggtg ttctgggcca gggatcagat gtgagccact gtttgaccta cactgcagct 26220
atggcaacgc tggatccttt aatcccttgt gtggtgccag ggactgaacc tgcgttctgg 26280
cactgcagag agagatgctg ctgatcttac tcagccacag caggaacccc taggatttaa 26340
taatttattc aaaataaacc aatttttctt attatataaa tactatatat agatactata 26400
agtaatattt tttaagaata gatcattggc ttaaaaaatg acatgttcag cccacaacta 26460
caccatctat gtttaaaaat gctaagaaga tgaagttaat agaaactata aggaaaacct 26520
actgaagtat cttaaatgat aaattatata acatatggct ggcagatcat acatattagt 26580
ggagaaactt tacaaaaagc cataaacagc tgatgctctt gaatgatgaa aagtaaaaat 26640
tcatggtagt gctgctttat aacaatatag taacttgtta agatttatgc tcagacatga 26700
agtctgttaa taccacattg gtagcactgt atgtgtgcct cacaatagat gagttttcaa 26760
acatggttgg gcttgttatg ttgcttttac ctgtctggca ttagcagcta gtcagactct 26820
taacatgaag tgcgacaaac acaacatgtc ctattaaata actttaaaat cttaacggtt 26880
tatcctgacc ttttcactaa tggtgaaacg caaacgtggg taaaactgcc aggacttggc 26940
agaaagaaag acagtggcac aaaactagca gtcattttct gccatccatt tgcagaatag 27000
ggggtggcag tttcacttaa agaatgtcct tgtgtgggag ttcccgtcgt ggttcagtgg 27060
ttaatgaatc cgactaggta ccgtgaggtt gcaggtttga tccctggcct tgctcagtgg 27120
gttaaggatc ccgagttgct gtggctctgg cgtaggccgg tggctacggc tccaattcag 27180
cccctagcct gggaacctcc atattgctga gggagtggcc caagaaatgg caaaaggaaa 27240
aaaaaaaggt ccttgtgaaa acaaattatt tgattaaatc tggaccgtga gtacataata 27300
cacatatggg tgatgaaatg gaaagtttgt ggtacttcat atgctaaaaa gtataatggt 27360
tgtctcagga aaaaacattc aagacagttg agttgcaagc tgatttagct cctattttct 27420
tggaatacta attttacttg aaagattgac tgtcaaattt tgattgtcac agttggatat 27480
ttggcagaca ttttttcaaa tacgaaggaa gttagccttt caaggaaaac tgacagcagt 27540
tgttgtcaat gataaaattc aaacttggaa gtgaaaatac agaattctgg ggaaacatct 27600
attagcaaga ggctgactat cagtactgtc cctagtgatc ggttgcttgc aaaagcagta 27660
attggaaagc actctcaaag aatccaagct tctgacctct tctcttcctc ccacagggcc 27720
tcgagacaat tgcatcgatg gtaccagtga aagtctgcaa tgaattcgag ttggcgcgcc 27780
tgtcattcta aatctctctt tcagcctaaa gctttttccc cgtatccccc caggtgtctg 27840
caggctcaaa gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt 27900
gcccgggctg tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc 27960
ggagccccgg gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg 28020
gggctttggg agggggctgt ccccgtgagc tcccaggcgc gcctgtcatt ctaaatctct 28080
ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc 28140
gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc 28200
acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc 28260
gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc 28320
tgtccccgtg agctcccagg cgcgccaact cggtaccgta tcgatgtcga cgttaacgct 28380
agtgatatca actttgtata gaaaagttgg ctccgaattt ctcgaggaat tccaattgac 28440
catgaccggt ttgtcgacaa gcttaattct accgggtagg ggaggcgctt ttcccaaggc 28500
agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct 28560
ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc 28620
ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg 28680
cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca 28740
gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct 28800
ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg 28860
ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc 28920
aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacccggg 28980
taactgatca taattcgacc caagtttgta caaaaaagca ggctgattac cggagaattc 29040
gccaccatgg atcctgatga tgttgttgat tcttctaaat cttttgtgat ggaaaacttt 29100
tcttcgtacc acgggactaa acctggttat gtagattcca ttcaaaaagg tatacaaaag 29160
ccaaaatctg gtacacaagg aaattatgac gatgattgga aagggtttta tagtaccgac 29220
aataaatacg acgctgcggg atactctgta gataatgaaa acccgctctc tggaaaagct 29280
ggaggcgtgg tcaaagtgac gtatccagga ctgacgaagg ttctcgcact aaaagtggat 29340
aatgccgaaa ctattaagaa agagttaggt ttaagtctca ctgaaccgtt gatggagcaa 29400
gtcggaacgg aagagtttat caaaaggttc ggtgatggtg cttcgcgtgt agtgctcagc 29460
cttcccttcg ctgaggggag ttctagcgtt gaatatatta ataactggga acaggcgaaa 29520
gcgttaagcg tagaacttga gattaatttt gaaacccgtg gaaaacgtgg ccaagatgcg 29580
atgtatgagt atatggctca agcctgtgca ggaaatcgtg tcaggcgatc tctttgtgaa 29640
ggaaccttac ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc 29700
actagttaag ggcccggcgc gcctaaggta cccccgggta actgaaaatc caccggatct 29760
agataactga tctacccagc tttcttgtac aaagtggtac gcgtgaattc actcctcagg 29820
tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc 29880
actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc 29940
tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt 30000
gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt 30060
tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat 30120
aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag 30180
ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat 30240
ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc 30300
cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggcg 30360
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 30420
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 30480
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc 30540
gacagtacta agcttggtag ataagttgat ttagccataa aatattgttt ccgtgacccc 30600
caattgcatc gatggtacca gtgaaagtct gcaatgaatt cgagttggcg cgcctgtcat 30660
tctaaatctc tctttcagcc taaagctttt tccccgtatc cccccaggtg tctgcaggct 30720
caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg 30780
gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc 30840
ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt 30900
tgggaggggg ctgtccccgt gagctcccag gcgcgcctgt cattctaaat ctctctttca 30960
gcctaaagct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag 31020
cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg 31080
ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct 31140
gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc 31200
cgtgagctcc caggcgcgcc aactcggtac cgtatcgatg tcgacggtgc gtttttatgc 31260
ttgtagtatt gtataatgtt tttaagatcc tagggataac agggtaatcg gccgctacta 31320
gggacaggat tggtgacaga aaagccccat ccttaggcct cctccttcct agtctcctga 31380
tattgggtct aacccccacc tcctgttagg cagattcctt atctggtgac acacccccat 31440
ttcctggagc catctctctc cttgccagaa cctctaaggt ttgcttacga tggagccaga 31500
gaggatcctg ggagggagag cttggcaggg ggtgggaggg aaggggggga tgcgtgacct 31560
gcccggttct cagtggccac cctgcgctac cctctcccag aacctgagct gctctgacgc 31620
ggctgtctgg tgcgtttcac tgatcctggt gctgcagctt ccttacactt cccaagagga 31680
gaagcagttt ggaaaaacaa aatcagaata agttggtcct gagttctaac tttggctctt 31740
cacctttcta gtccccaatt tatattgttc ctccgtgcgt cagttttacc tgtgagataa 31800
ggccagtagc cagccccgtc ctggcagggc tgtggtgagg aggggggtgt ccgtgtggaa 31860
aactcccttt gtgagaatgg tgcgtcctag gtgttcacca ggtcgtggcc gcctctactc 31920
cctttctctt tctccatcct tctttcctta aagagtcccc agtgctatct gggacatatt 31980
cctccgccca gagcagggtc ccgcttccct aaggccctgc tctgggcttc tgggtttgag 32040
tccttggcaa gcccaggaga ggcgctcagg cttccctgtc ccccttcctc gtccaccatc 32100
tcatgcccct ggctctcctg ccccttccct acaggggttc ctggctctgc tctccaccgc 32160
gggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaataata 32220
tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc 32280
catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat 32340
gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat 32400
gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 32460
gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 32520
aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa 32580
acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 32640
gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 32700
cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 32760
gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 32820
cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 32880
atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 32940
cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 33000
aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 33060
ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc 33120
attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac 33180
gcgtcgttcc actgagcgtc agac 33204
Claims (10)
1. The homologous recombination vector for the mammary gland specific expression of the antibacterial peptide Cecropin gene is characterized in that the homologous recombination vector takes a eukaryotic expression vector as a skeleton vector, and at least comprises a positive screening element, a negative screening marker gene, an antibacterial peptide Cecropin gene expression box, and a homologous left arm and a homologous right arm of a rosa26 gene;
wherein the positive screening element comprises a recombinase recognition sequence-positive screening marker gene-recombinase recognition sequence connected in sequence;
one end of the positive screening element is connected with the homologous left arm, and the other end of the positive screening element is connected with the antibacterial peptide Cecropin gene expression cassette; the other end of the antibacterial peptide Cecropin gene expression box is connected with the homologous right arm, and the outer side of the homologous right arm is connected with a negative screening marker gene.
2. The homologous recombinant vector according to claim 1, wherein the positive selection marker gene comprises a gene encoding neomycin phosphotransferase, hygromycin B phosphotransferase, hypoxanthine phosphoribosyltransferase or puromycin acetyltransferase; the negative selection marker gene comprises a gene encoding diphtheria toxin or thymidine; the recombinase gene includes a gene encoding Cre enzyme or Flp enzyme.
3. The homologous recombination vector of claim 2, wherein the positive selection element is Loxp-OCT4 promoter-Cre-PGK-Neo-Loxp.
4. The homologous recombinant vector according to claim 1, wherein the expression cassette of Cecropin gene is hLF promoter-Cecropin gene-hLF terminator.
5. The homologous recombination vector according to claim 1, wherein said homologous recombination vector comprises the following elements linked in sequence: homologous left arm-Loxp-OCT 4 promoter-Cre-PGK-Neo-Loxp-human lactoferrin hLF promoter-antibacterial peptide Cecropin gene-hLF terminator-homologous right arm-PGK-dTA.
6. The homologous recombination vector of claim 5, wherein the homologous left arm and the homologous right arm are the homologous left arm and the homologous right arm from the porcine rosa26 gene, and the nucleotide sequences thereof are shown in SEQ ID NO 1 and 2, respectively; the nucleotide sequence of the antibacterial peptide Cecropin gene is shown in SEQ ID NO. 3.
7. The homologous recombination vector of claim 1, wherein the nucleotide sequence of the homologous recombination vector is represented by SEQ ID NO. 4.
8. A cell line specifically expressing Cecropin gene, which is characterized in that the homologous recombination vector of any one of claims 1 to 7 is used to transfect mammalian somatic cells to obtain a target-positive cell clone, namely the cell line specifically expressing Cecropin gene.
9. Use of the homologous recombination vector of any one of claims 1 to 7 or the cell line of claim 8 for the preparation of a transgenic animal for the mammary gland-specific expression of Cecropin, wherein the animal comprises a pig.
10. Use according to claim 9, characterized in that the homologous recombination vector is transfected into the same porcine cell line as the source of the DNA of its homology arms.
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