CN114107225B - SARS-CoV-2 adaptive strain based on KI-hACE2 mouse and its construction method - Google Patents

SARS-CoV-2 adaptive strain based on KI-hACE2 mouse and its construction method Download PDF

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CN114107225B
CN114107225B CN202210083596.0A CN202210083596A CN114107225B CN 114107225 B CN114107225 B CN 114107225B CN 202210083596 A CN202210083596 A CN 202210083596A CN 114107225 B CN114107225 B CN 114107225B
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石正丽
刘美琴
林浩枫
蒋人地
周鹏
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Jiangsu Ningpu Medical Technology Co ltd
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Nanjing Keguan Biomedical Technology Co ltd
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Abstract

The invention relates to the technical field of coronaviruses, in particular to a SARS-CoV-2 adaptive strain based on KI-hACE2 mice and a construction method thereof, wherein the full-length genome sequence of the adaptive strain is shown as SEQ ID NO. 1. SARS-CoV-2 adapted strain based on KI-hACE2 mouse can produce typical clinical symptoms of respiratory distress, piloerection, hunch back, weight loss and even death, and can produce lung pathological changes such as pneumonia, inflammatory cell infiltration and the like; the method is characterized in that continuous passage adaptation is carried out on the basis of human ACE2, which is closer to the propagation adaptation of viruses in human groups; the pathological phenomenon of the clinical COVID-19 patient is well simulated, and a good evaluation platform is provided for the research and development of antiviral drugs and vaccines.

Description

SARS-CoV-2 adaptive strain based on KI-hACE2 mouse and its construction method
Technical Field
The invention relates to the technical field of coronavirus, in particular to a SARS-CoV-2 adaptive strain based on KI-hACE2 mouse.
Background
The outbreak of COVID-19 has caused a serious harm to the human health and economic development in the world, and 236,599,025 cases have been diagnosed globally by 10, 9 months in 2021, wherein 4,831,486 cases die.
Patients infected with SARS-CoV-2 may develop fever, fatigue, dry cough, mild pneumonia or severe acute respiratory distress syndrome. The good animal model can provide an evaluation platform for the development of antiviral drugs and the development of vaccines, and the virus can be quickly eliminated after the mice are infected with SARS-CoV-2, so that the virus can not be replicated in the mice, and the development of the good mouse model is particularly important.
At present, the existing SARS-CoV-2 mouse models comprise a hACE2 transgenic mouse model, an AAV-hACE2 transduced mouse model, an Ad5-hACE2 transduced mouse model and a wild type mouse adapted strain, and the mouse models can make the virus well replicate but can not simulate the symptoms of clinical patients.
Therefore, it is very necessary to construct a mouse adapted strain that can simulate the symptoms of clinical patients, which will provide a good evaluation platform for the development of antiviral drugs and vaccines.
Disclosure of Invention
The invention aims to provide a SARS-CoV-2 adaptive strain based on KI-hACE2 mouse and a construction method thereof. The adaptive strain integrates the advantages of the existing mouse model, can generate clinical symptoms such as typical respiratory distress, piloerection, hunch back, weight loss and even death, and can generate lung pathological changes such as pneumonia, inflammatory cell infiltration and the like. The adapted strain is not like a wild-type mouse (the virus is adapted by passage on the basis of mouse ACE 2), but is adapted by continuous passage on the basis of human ACE2, and the adaptation is closer to the transmission adaptation of the virus in a human population.
In order to achieve the purpose, the invention provides the following technical scheme:
a SARS-CoV-2 adaptive strain based on KI-hACE2 mouse has the full-length genome sequence shown in SEQ ID NO. 1.
The method for constructing the SARS-CoV-2 adaptive strain based on KI-hACE2 mouse comprises the following steps:
(1) SARS-CoV-2(GenBank: MN996528.1), was isolated from the alveolar lavage fluid of patients with COVID-19. The sequence is shown as SEQ ID NO: 2, respectively.
(2) The expression cassette of hACE2-Wpre-pA is knocked in the starting codon site of the Ace2 gene in a site-specific mode by using CRISPR/Cas9 technology through homologous recombination.
The brief procedure is as follows: obtaining Cas9 mRNA and gRNA through an in vitro transcription mode; a homologous recombinant vector (donor vector) was constructed by the method of In-Fusion cloning, which contained a 3.0 kb 5 'homology arm, hACE2-Wpre-pA, and a 3.0 kb 3' homology arm. Cas9 mRNA, gRNA, and donor vector were microinjected into fertilized eggs of C57BL/6J mice to obtain F0-generation mice. The F0 mice positive for PCR amplification and sequencing identification were mated with C57BL/6J mice to obtain 4 positive F1 mice.
(3) Obtaining SARS-CoV-2 adapted strain based on KI-hACE2 mouse
a. A50 microliter drop of the virus stock solution cultured in VeroE6 cells was infected with 4 KI-hACE2 mice, i.e., offspring mice bred at F1 generation, which were half-female.
b. Infected mice were kept on a constant rearing basis, and were weighed daily and observed for mental status. Dissections were performed 2-3 days after infection, and all lung tissue of mice was ground using a grinding tube containing DMEM medium. And taking 140 mul of supernatant, adding the supernatant into 560 mul of AVL cracking Buffer, standing at room temperature for 10min, and adding 560 mul of absolute ethyl alcohol.
c. Nucleic acid is extracted by using a nucleic acid extracting reagent QIAamp Viral RNA Mini kit (QIAGEN), quantification is carried out, and the lung tissue with the highest virus content is tested.
Quantitative reagent: HiSxript II One step qRT-PCR SYBR Green Kit (Vazyme);
and (3) quantitative primer pair: RBD-qF1: 5'-CAATGGTTTAACAGGCACAGG-3';
RBD-qR1: 5'-CTCAAGTGTCTGTGGATCACG-3'. As shown in SEQ ID NO: 3-4.
The reaction system is as follows: 2X OneStep SYBR Green mix 5 mu l, 0.5 mu l of OneStep SYBR Green Enzyme mix, 0.2 mu l of 50X ROX Dye10.2 mu l of RBD-qF1 and 0.2 mu l of RBD-qR1 respectively, 1.9 mu l of water without nuclease and 2 mu l of templates respectively.
d. One lung tissue with the highest virus content is taken, centrifuged for 10min at 10000g and 4 ℃, and 50 mu l of supernatant is taken to be dripped into the nose to infect the other 4 mice.
e. And circulating the steps a-d until the mice have obvious clinical symptoms such as weight loss, respiratory distress and the like.
Compared with the prior art, the invention has the beneficial effects that:
the SARS-CoV-2 adaptive strain based on KI-hACE2 mouse is passed in mouse with knocked-in human ACE2 gene, so that SARS-CoV-2 can adapt to human ACE2 continuously in continuous passage process, the phenomenon of virus spreading in natural population is simulated well, and the mice infected with non-pathogenic virus have obvious clinical symptoms of respiratory distress, arch back, pilus, weight reduction and even death. The lung tissue has obvious pathological changes, and pathological phenomena such as mononuclear lymphocytes and other inflammatory cell infiltration, alveolar wall thickening, alveolar structure destruction, cellulose exudation and the like appear. The model well simulates the pathological phenomena of the COVID-19 clinical patient and provides a good evaluation platform for the research and development of antiviral drugs and vaccines.
Drawings
FIG. 1 is a schematic diagram of the construction strategy of SARS-CoV-2 adapted strain based on KI-hACE2 mouse according to the present invention;
FIG. 2 shows the body weight change of SARS-CoV-2 infected KI-hACE2 mice;
FIG. 3 shows the tissue viral load of SARS-CoV-2 infected KI-hACE2 mouse;
FIG. 4 shows the pathological changes of lung tissues of SARS-CoV-2-infected KI-hACE2 mice;
FIG. 5 shows the body weight changes of mice infected with KI-hACE2 with SARS-CoV-2 adapted strain from different clones ( clones 1, 3, 5, 8, 9, 11);
FIG. 6 shows the number of copies of the virus in each tissue (each upper triangle, lower triangle, dot, square, etc. in the figure represents a mouse);
FIG. 7 shows the pathological changes of different clone SARS-CoV-2 adapted strain infected with KI-hACE 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
As shown in FIG. 1, the method for constructing SARS-CoV-2 adapted strain based on KI-hACE2 mouse comprises the following steps:
(1) SARS-CoV-2(GenBank: MN996528.1), was isolated from the alveolar lavage fluid of patients with COVID-19. The sequence is shown as SEQ ID NO: 2, respectively.
(2) The expression cassette of hACE2-Wpre-pA is knocked in the starting codon site of the Ace2 gene in a site-specific mode by using CRISPR/Cas9 technology through homologous recombination.
The brief procedure is as follows: obtaining Cas9 mRNA and gRNA through an in vitro transcription mode; a homologous recombinant vector (donor vector) was constructed by the method of In-Fusion cloning, which contained a 3.0 kb 5 'homology arm, hACE2-Wpre-pA, and a 3.0 kb 3' homology arm. Cas9 mRNA, gRNA, and donor vector were microinjected into fertilized eggs of C57BL/6J mice to obtain F0-generation mice. The F0 mice positive for PCR amplification and sequencing identification were mated with C57BL/6J mice to obtain 4 positive F1 mice.
The mouse model was constructed by Shanghai's Square model Biotechnology, Inc.
(3) Obtaining SARS-CoV-2 adapted strain based on KI-hACE2 mouse
a. A50. mu.l drop of virus stock cultured in VeroE6 cells was infected with 4 KI-hACE2 mice, half male and half female.
b. Infected mice were kept on a constant rearing basis, and were weighed daily and observed for mental status. Dissections were performed 2-3 days after infection, and all lung tissue of mice was ground using a grinding tube containing DMEM medium. And taking 140 mul of supernatant, adding the supernatant into 560 mul of AVL cracking Buffer, standing at room temperature for 10min, and adding 560 mul of absolute ethyl alcohol.
c. Nucleic acid is extracted by using a nucleic acid extracting reagent QIAamp Viral RNA Mini kit (QIAGEN), quantification is carried out, and the lung tissue with the highest virus content is tested.
Quantitative reagent: HiSxript II One step qRT-PCR SYBR Green Kit (Vazyme);
and (3) quantitative primer pair: RBD-qF1: 5'-CAATGGTTTAACAGGCACAGG-3';
RBD-qR1: 5'-CTCAAGTGTCTGTGGATCACG-3'. As shown in SEQ ID NO: 3-4.
The reaction system is as follows: 2X OneStep SYBR Green mix 5 mu l, 0.5 mu l of OneStep SYBR Green Enzyme mix, 0.2 mu l of 50X ROX Dye10.2 mu l of RBD-qF1 and 0.2 mu l of RBD-qR1 respectively, 1.9 mu l of water without nuclease and 2 mu l of templates respectively.
d. One lung tissue with the highest virus content is taken, centrifuged for 10min at 10000g and 4 ℃, and 50 mu l of supernatant is taken to be dripped into the nose to infect the other 4 mice.
e. And circulating the steps a-d until the mice have obvious clinical symptoms such as weight loss, respiratory distress and the like.
The full-length genome sequence of the SARS-CoV-2 adapted strain based on KI-hACE2 mouse is shown in SEQ ID NO. 1.
EXAMPLE 2 evaluation of SARS-CoV-2 adapted Strain based on KI-hACE2 mice
(1) The lethally competent mouse lung tissue supernatant obtained in example 1 was infected with VeroE6 cells, 6 clones ( clones 1, 3, 5, 8, 9, 11) were selected by plaque purification and expanded in VeroE6 cells. 2x 10 for each clone4TCID50 virus infected 8-month-old KI-hACE2 mice, 8 mice per clone. Weighing is performed daily and clinical symptoms are observed until day 14 post-infection. The results showed that clone 9 was most lethal to mice, with a mortality rate of 62.5% (5/8), clone 8 was least effective in infection, and mice gradually recovered after weight loss, and no mice died, as shown in fig. 5.
(2) Several mice infected and dead are selected for each clone, tissues such as heart, liver, spleen, lung, kidney, intestine, brain, turbinate and the like are taken, the tissues are ground in a cell culture medium, and the supernatant is taken to detect the virus copy number in each tissue. There was no difference in viral replication between different clones, the viral content was higher in respiratory tissues (lung, turbinate, trachea), and the viral copy number could be detected in individual mice in other tissues as shown in fig. 6.
(3) A part of lung tissue of a mouse was fixed with a tissue fixing solution, and the degree of lesion of lung tissue was observed by a tissue virus section, for example, clone 9, as shown in FIG. 7. The 1 st graph shows that HE staining shows that lung tissues have obvious pneumonia phenomena such as inflammatory cell infiltration and alveolar wall thickening after KI-hACE2 mice are infected with clone 9 virus strain; FIG. 2 shows that the staining of Masson shows that there is a large amount of cellulose exudation in lung tissue and fibrosis in the lung; the 3 rd panel shows that the lung tissue sections were immunofluorescent to detect the presence of significant virus, indicating that there was a significant amount of virus replication in the lung tissue.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
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<120> SARS-CoV-2 adapted strain based on KI-hACE2 mouse and its construction method
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ataccttccc aggtaacaaa ccaaccaact ttcgatctct tgtagatctg ttctctaaac 60
gaactttaaa atctgtgtgg ctgtcactcg gctgcatgct tagtgcactc acgcagtata 120
attaataact aattactgtc gttgacagga cacgagtaac tcgtctatct tctgcaggct 180
gcttacggtt tcgtccgtgt tgcagccgat catcagcaca tctaggtttc gtccgggtgt 240
gaccgaaagg taagatggag agccttgtcc ctggtttcaa cgagaaaaca cacgtccaac 300
tcagtttgcc tgttttacag gttcgcgacg tgctcgtacg tggctttgga gactccgtgg 360
aggaggtctt atcagaggca cgtcaacatc ttaaagatgg cacttgtggc ttagtagaag 420
ttgaaaaagg cgttttgcct caacttgaac agccctatgt gttcatcaaa cgttcggatg 480
ctcgaactgc acctcatggt catgttatgg ttgagctggt agcagaactc gaaggcattc 540
agtacggtcg tagcggtgag acacttggtg tccttgtccc tcatgtgggc gaaataccag 600
tggcttaccg caaggttctt cttcgtaaga acggtaataa aggagctggt ggccatagtt 660
acggcgccga tctaaagtca tttgacttag gcgacgagct tggcactgat ccttatgaag 720
attttcaaga aaactggaac actaaacata gcagtggtgt tacccgtgaa ctcatgcgtg 780
agcttaacgg aggggcatac actcgctatg tcgataacaa cttctgtggc cctgatggct 840
accctcttga gtgcattaaa gaccttctag cacgtgctgg taaagcttca tgcactttgt 900
ccgaacaact ggactttatt gacactaaga ggggtgtata ctgctgccgt gaacatgagc 960
atgaaattgc ttggtacacg gaacgttctg aaaagagcta tgaattgcag acaccttttg 1020
aaattaaatt ggcaaagaaa tttgacacct tcaatgggga atgtccaaat tttgtatttc 1080
ccttaaattc cataatcaag actattcaac caagggttga aaagaaaaag cttgatggct 1140
ttatgggtag aattcgatct gtctatccag ttgcgtcacc aaatgaatgc aaccaaatgt 1200
gcctttcaac tctcatgaag tgtgatcatt gtggtgaaac ttcatggcag acgggcgatt 1260
ttgttaaagc cacttgcgaa ttttgtggca ctgagaattt gactaaagaa ggtgccacta 1320
cttgtggtta cttaccccaa aatgctgttg ttaaaattta ttgtccagca tgtcacaatt 1380
cagaagtagg acctgagcat agtcttgccg aataccataa tgaatctggc ttgaaaacca 1440
ttcttcgtaa gggtggtcgc actattgcct ttggaggctg tgtgttctct tatgttggtt 1500
gccataacaa gtgtgcctat tgggttccac gtgctagcgc taacataggt tgtaaccata 1560
caggtgttgt tggagaaggt tccgaaggtc ttaatgacaa ccttcttgaa atactccaaa 1620
aagagaaagt caacatcaat attgttggtg actttaaact taatgaagag atcgccatta 1680
ttttggcatc tttttctgct tccacaagtg ctttagtgga aactgtgaaa ggtttggatt 1740
ataaagcatt caaacaaatt gttgaatcct gtggtaattt taaagttaca aaaggaaaag 1800
ctaaaaaagg tgcctggaat attggtgaac agaaatcaat actgagtcct ctttatgcat 1860
ttgcatcaga ggctgctcgt gttgtacgat caattttctc ccgcactctt gaaactgctc 1920
aaaattctgt gcgtgtttta cagaaggccg ctataacaat actagatgga atttcacagt 1980
attcactgag actcattgat gctatgatgt tcacatctga tttggctact aacaatctag 2040
ttgtaatggc ctacattaca ggtggtgttg ttcagttgac ttcgcagtgg ctaactaaca 2100
tctttggcac tgtttatgaa aaactcaaac ccgtccttga ttggcttgaa gagaagttta 2160
aggaaggtgt agagtttctt agagacggtt gggaaattgt taaatttatc tcaacctgtg 2220
cttgtgaaat tgtcggtgga caaattgtca cctgtgcaaa ggaaattaag gagagtgttc 2280
agacattctt taagcttgta aataaatttt tggctttgtg tgctgactct atcattattg 2340
gtggagctaa acttaaagcc ttgaatttag gtgaaacatt tgtcacgcac tcaaagggat 2400
tgtacagaaa gtgtgttaaa tccagagaag aaactggcct actcatgcct ctaaaagccc 2460
caaaagaaat tatcttctta gagggagaaa cacttcccac agaagtgtta acagaggaag 2520
ttgtcttgaa aactggtgat ttacaaccat tagaacaacc tactagtgaa gctgttgaag 2580
ctccattggt tggtacacca gtttgtatta acgggcttat gttgctcgaa atcaaagaca 2640
cagaaaagta ctgtgccctt gcacctaata tgatggtaac aaacaatacc ttcacactca 2700
aaggcggtgc accaacaaag gttacttttg gtgatgacac tgtgatagaa gtgcaaggtt 2760
acaagagtgt gaatatcact tttgaacttg atgaaaggat tgataaagta cttaatgaga 2820
agtgctctgc ctatacagtt gaactcggta cagaagtaaa tgagttcgcc tgtgttgtgg 2880
cagatgctgt cataaaaact ttgcaaccag tatctgaatt acttacacca ctgggcattg 2940
atttagatga gtggagtatg gctacatact acttatttga tgagtctggt gagtttaaat 3000
tggcttcaca tatgtattgt tctttctacc ctccagatga ggatgaagaa gaaggtgatt 3060
gtgaagaaga agagtttgag ccatcaactc aatatgagta tggtactgaa gatgattacc 3120
aaggtaaacc tttggaattt ggtgccactt ctgctgctct tcaacctgaa gaagagcaag 3180
aagaagattg gttagatgat gatagtcaac aaactgttgg tcaacaagac ggcagtgagg 3240
acaatcagac aactactatt caaacaattg ttgaggttca acctcaatta gagatggaac 3300
ttacaccagt tgttcagact attgaagtga atagttttag tggttattta aaacttactg 3360
acaatgtata cattaaaaat gcagacattg tggaagaagc taaaaaggta aaaccaacag 3420
tggttgttaa tgcagccaat gtttacctta aacatggagg aggtgttgca ggagccttaa 3480
ataaggctac taacaatgcc atgcaagttg aatctgatga ttacatagct actaatggac 3540
cacttaaagt gggtggtagt tgtgttttaa gcggacacaa tcttgctaaa cactgtcttc 3600
atgttgtcgg cccaaatgtt aacaaaggtg aagacattca acttcttaag agtgcttatg 3660
aaaattttaa tcagcacgaa gttctacttg caccattatt atcagctggt atttttggtg 3720
ctgaccctat acattcttta agagtttgtg tagatactgt tcgcacaaat gtctacttag 3780
ctgtctttga taaaaatctc tatgacaaac ttgtttcaag ctttttggaa atgaagagtg 3840
aaaagcaagt tgaacaaaag atcgctgaga ttcctaaaga ggaagttaag ccatttataa 3900
ctgaaagtaa accttcagtt gaacagagaa aacaagatga taagaaaatc aaagcttgtg 3960
ttgaagaagt tacaacaact ctggaagaaa ctaagttcct cacagaaaac ttgttacttt 4020
atattgacat taatggcaat cttcatccag attctgccac tcttgttagt gacattgaca 4080
tcactttctt aaagaaagat gctccatata tagtgggtga tgttgttcaa gagggtgttt 4140
taactgctgt ggttatacct actaaaaagg ctggtggcac tactgaaatg ctagcgaaag 4200
ctttgagaaa agtgccaaca gacaattata taaccactta cccgggtcag ggtttaaatg 4260
gttacactgt agaggaggca aagacagtgc ttaaaaagtg taaaagtgcc ttttacattc 4320
taccatctat tatctctaat gagaagcaag aaattcttgg aactgtttct tggaatttgc 4380
gagaaatgct tgcacatgca gaagaaacac gcaaattaat gcctgtctgt gtggaaacta 4440
aagccatagt ttcaactata cagcgtaaat ataagggtat taaaatacaa gagggtgtgg 4500
ttgattatgg tgctagattt tacttttaca ccagtaaaac aactgtagcg tcacttatca 4560
acacacttaa cgatctaaat gaaactcttg ttacaatgcc acttggctat gtaacacatg 4620
gcttaaattt ggaagaagct gctcggtata tgagatctct caaagtgcca gctacagttt 4680
ctgtttcttc acctgatgct gttacagcgt ataatggtta tcttacttct tcttctaaaa 4740
cacctgaaga acattttatt gaaaccatct cacttgctgg ttcctataaa gattggtcct 4800
attctggaca atctacacaa ctaggtatag aatttcttaa gagaggtgat aaaagtgtat 4860
attacactag taatcctacc acattccacc tagatggtga agttatcacc tttgacaatc 4920
ttaagacact tctttctttg agagaagtga ggactattaa ggtgtttaca acagtagaca 4980
acattaacct ccacacgcaa gttgtggaca tgtcaatgac atatggacaa cagtttggtc 5040
caacttattt ggatggagct gatgttacta aaataaaacc tcataattca catgaaggta 5100
aaacatttta tgttttacct aatgatgaca ctctacgtgt tgaggctttt gagtactacc 5160
acacaactga tcctagtttt ctgggtaggt acatgtcagc attaaatcac actaaaaagt 5220
ggaaataccc acaagttaat ggtttaactt ctattaaatg ggcagataac aactgttatc 5280
ttgccactgc attgttaaca ctccaacaaa tagagttgaa gtttaatcca cctgctctac 5340
aagatgctta ttacagagca agggctggtg aagctgctaa cttttgtgca cttatcttag 5400
cctactgtaa taagacagta ggtgagttag gtgatgttag agaaacaatg agttacttgt 5460
ttcaacatgc caatttagat tcttgcaaaa gagtcttgaa cgtggtgtgt aaaacttgtg 5520
gacaacagca gacaaccctt aagggtgtag aagctgttat gtacatgggc acactttctt 5580
atgaacaatt taagaaaggt gttcagatac cttgtacgtg tggtaaacaa gctacaaaat 5640
atctagtaca acaggagtca ccttttgtta tgatgtcagc accacctgct cagtatgaac 5700
ttaagcatgg tacatttact tgtgctagtg agtacactgg taattaccag tgtggtcact 5760
ataaacatat aacttctaaa gaaactttgt attgcataga cggtgcttta cttacaaagt 5820
cctcagaata caaaggtcct attacggatg ttttctacaa agaaaacagt tacacaacaa 5880
ccataaaacc agttacttat aaattggatg gtgttgtttg tacagaaatt gaccctaagt 5940
tggacaatta ttataagaaa gacaattctt atttcacaga gcaaccaatt gatcttgtac 6000
caaaccaacc atatccaaac gcaagcttcg ataattttaa gtttgtatgt gataatatca 6060
aatttgctga tgatttaaac cagttaactg gttataagaa acctgcttca agagagctta 6120
aagttacatt tttccctgac ttaaatggtg atgtggtggc tattgattat aaacactaca 6180
caccctcttt taagaaagga gctaaattgt tacataaacc tattgtttgg catgttaaca 6240
atgcaactaa taaagccacg tataaaccaa atacctggtg tatacgttgt ctttggagca 6300
caaaaccagt tgaaacatca aattcgtttg atgtactgaa gtcagaggac gcgcagggaa 6360
tggataatct tgcctgcgaa gatctaaaac cagtctctga agaagtagtg gaaaatccta 6420
ccatacagaa agacgttctt gagtgtaatg tgaaaactac cgaagttgta ggagacatta 6480
tacttaaacc agcaaataat agtttaaaaa ttacagaaga ggttggccac acagatctaa 6540
tggctgctta tgtagacaat tctagtctta ctattaagaa acctaatgaa ttatctagag 6600
tattaggttt gaaaaccctt gctactcatg gtttagctgc tgttaatagt gtcccttggg 6660
atactatagc taattatgct aagccttttc ttaacaaagt tgttagtaca actactaaca 6720
tagttacacg gtgtttaaac cgtgtttgta ctaattatat gccttatttc tttactttat 6780
tgctacaatt gtgtactttt actagaagta caaattctag aattaaagca tctatgccga 6840
ctactatagc aaagaatact gttaagagtg tcggtaaatt ttgtctagag gcttcattta 6900
attatttgaa gtcacctaat ttttctaaac tgataaatat tataatttgg tttttactat 6960
taagtgtttg cctaggttct ttaatctact caaccgctgc tttaggtgtt ttaatgtcta 7020
atttaggcat gccttcttac tgtactggtt acagagaagg ctatttgaac tctactaatg 7080
tcactattgc aacctactgt actggttcta taccttgtag tgtttgtctt agtggtttag 7140
attctttaga cacctatcct tctttagaaa ctatacaaat taccatttca tcttttaaat 7200
gggatttaac tgcttttggc ttagttgcag agtggttttt ggcatatatt cttttcacta 7260
ggtttttcta tgtacttgga ttggctgcaa tcatgcaatt gtttttcagc tattttgcag 7320
tacattttat tagtaattct tggcttatgt ggttaataat taatcttgta caaatggccc 7380
cgatttcagc tatggttaga atgtacatct tctttgcatc attttattat gtatggaaaa 7440
gttatgtgca tgttgtagac ggttgtaatt catcaacttg tatgatgtgt tacaaacgta 7500
atagagcaac aagagtcgaa tgtacaacta ttgttaatgg tgttagaagg tccttttatg 7560
tctatgctaa tggaggtaaa ggcttttgca aactacacaa ttggaattgt gttaattgtg 7620
atacattctg tgctggtagt acatttatta gtgatgaagt tgcgagagac ttgtcactac 7680
agtttaaaag accaataaat cctactgacc agtcttctta catcgttgat agtgttacag 7740
tgaagaatgg ttccatccat ctttactttg ataaagctgg tcaaaagact tatgaaagac 7800
attctctctc tcattttgtt aacttagaca acctgagagc taataacact aaaggttcat 7860
tgcctattaa tgttatagtt tttgatggta aatcaaaatg tgaagaatca tctgcaaaat 7920
cagcgtctgt ttactacagt cagcttatgt gtcaacctat actgttacta gatcaggcat 7980
tagtgtctga tgttggtgat agtgcggaag ttgcagttaa aatgtttgat gcttacgtta 8040
atacgttttc atcaactttt aacgtaccaa tggaaaaact caaaacacta gttgcaactg 8100
cagaagctga acttgcaaag aatgtgtcct tagacaatgt cttatctact tttatttcag 8160
cagctcggca agggtttgtt gattcagatg tagaaactaa agatgttgtt gaatgtctta 8220
aattgtcaca tcaatctgac atagaagtta ctggcgatag ttgtaataac tatatgctta 8280
cctataacaa agttgaaaac atgacacccc gtgaccttgg tgcttgtatt gactgtagtg 8340
cgcgtcatat taatgcgcag gtagcaaaaa gtcacaacat tgctttgata tggaacgtta 8400
aagatttcat gtcattgtct gaacaactac gaaaacaaat acgtagtgct gctaaaaaga 8460
ataacttacc ttttaagttg acatgtgcaa ctactagaca agttgttaat gttgtaacaa 8520
caaagatagc acttaagggt ggtaaaattg ttaataattg gttgaagcag ttaattaaag 8580
ttacacttgt gttccttttt gttgctgcta ttttctattt aataacacct gttcatgtca 8640
tgtctaaaca tactgacttt tcaagtgaaa tcataggata caaggctatt gatggtggtg 8700
tcactcgtga catagcatct acagatactt gttttgctaa caaacatgct gattttgaca 8760
catggtttag ccagcgtggt ggtagttata ctaatgacaa agcttgccca ttgattgctg 8820
cagtcataac aagagaagtg ggttttgtcg tgcctggttt gcctggcacg atattacgca 8880
caactaatgg tgactttttg catttcttac ctagagtttt tagtgcagtt ggtaacatct 8940
gttacacacc atcaaaactt atagagtaca ctgactttgc aacatcagct tgtgttttgg 9000
ctgctgaatg tacaattttt aaagatgctt ctggtaagcc agtaccatat tgttatgata 9060
ccaatgtact agaaggttct gttgcttatg aaagtttacg ccctgacaca cgttatgtgc 9120
tcatggatgg ctctattatt caatttccta acacctacct tgaaggttct gttagagtgg 9180
taacaacttt tgattctgag tactgtaggc acggcacttg tgaaagatca gaagctggtg 9240
tttgtgtatc tactagtggt agatgggtac ttaacaatga ttattacaga tctttaccag 9300
gagttttctg tggtgtagat gctgtaaatt tacttactaa tatgtttaca ccactaattc 9360
aacctattgg tgctttggac atatcagcat ctatagtagc tggtggtatt gtagctatcg 9420
tagtaacatg ccttgcctac tattttatga ggtttagaag agcttttggt gaatacagtc 9480
atgtagttgc ctttaatact ttactattcc ttatgtcatt cactgtactc tgtttaacac 9540
cagtttactc attcttacct ggtgtttatt ctgttattta cttgtacttg acattttatc 9600
ttactaatga tgtttctttt ttagcacata ttcagtggat ggttatgttc acacctttag 9660
tacctttctg gataacaatt gcttatatca tttgtatttc cacaaagcat ttctattggt 9720
tctttagtaa ttacctaaag agacgtgtag tctttaatgg tgtttccttt agtacttttg 9780
aagaagctgc gctgtgcacc tttttgttaa ataaagaaat gtatctaaag ttgcgtagtg 9840
atgtgctatt acctcttacg caatataata gatacttagc tctttataat aagtacaagt 9900
attttagtgg agcaatggat acaactagct acagagaagc tgcttgttgt tatctcgcaa 9960
aggctctcaa tgacttcagt aactcaggtt ctgatgttct ttaccaacca ccacaaacct 10020
ctatcacctc agctgttttg cagagtggtt ttagaaaaat ggcattccca tctggtaaag 10080
ttgagggttg tatggtacaa gtaacttgtg gtacaactac acttaacggt ctttggcttg 10140
atgacgtagt ttactgtcca agacatgtga tctgcacctc tgaagacatg cttaacccta 10200
attatgaaga tttactcatt cgtaagtcta atcataattt cttggtacag gctggtaatg 10260
ttcaactcag ggttattgga cattctatgc aaaattgtgt acttaagctt aaggttgata 10320
cagccaatcc taagacacct aagtataagt ttgttcgcat tcaaccagga cagacttttt 10380
cagtgttagc ttgttacaat ggttcaccat ctggtgttta ccaatgtgct atgaggccca 10440
atttcactat taagggttca ttccttaatg gttcatgtgg tagtgttggt tttaacatag 10500
attatgactg tgtctctttt tgttacatgc accatatgga attaccaact ggagttcatg 10560
ctggcacaga cttagaaggt aacttttatg gaccttttgt tgacaggcaa acagcacaag 10620
cagctggtac ggacacaact attacagtta atgttttagc ttggttgtac gctgctgtta 10680
taaatggaga caggtggttt ctcaatcgat ttaccacaac tcttaatgac tttaaccttg 10740
tggctatgaa gtacaattat gaacctctaa cacaagacca tgttgacata ctaggacctc 10800
tttctgctca aactggaatt gccgttttag atatgtgtgc ttcattaaaa gaattactgc 10860
aaaatggtat gaatggacgt accatattgg gtagtgcttt attagaagat gaatttacac 10920
cttttgatgt tgttagacaa tgctcaggtg ttactttcca aagtgcagtg aaaagaacaa 10980
tcaagggtac acaccactgg ttgttactca caattttgac ttcactttta gttttagtcc 11040
agagtactca atggtctttg ttcttttttt tgtatgaaaa tgccttttta ccttttgcta 11100
tgggtattat tgctatgtct gcttttgcaa tgatgtttgt caaacataag catgcatttc 11160
tctgtttgtt tttgttacct tctcttgcca ctgtagctta ttttaatatg gtctatatgc 11220
ctgctagttg ggtgatgcgt attatgacat ggttggatat ggttgatact agtttgtctg 11280
gttttaagct aaaagactgt gttatgtatg catcagctgt agtgttacta atccttatga 11340
cagcaagaac tgtgtatgat gatggtgcta ggagagtgtg gacacttatg aatgtcttga 11400
cactcgttta taaagtttat tatggtaatg ctttagatca agccatttcc atgtgggctc 11460
ttataatctc tgttacttct aactactcag gtgtagttac aactgtcatg tttttggcca 11520
gaggtattgt ttttatgtgt gttgagtatt gccctatttt cttcataact ggtaatacac 11580
ttcagtgtat aatgctagtt tattgtttct taggctattt ttgtacttgt tactttggcc 11640
tcttttgttt actcaaccgc tactttagac tgactcttgg tgtttatgat tacttagttt 11700
ctacacagga gtttagatat atgaattcac agggactact cccacccaag aatagcatag 11760
atgccttcaa actcaacatt aaattgttgg gtgttggtgg caaaccttgt atcaaagtag 11820
ccactgtaca gtctaaaatg tcagatgtaa agtgcacatc agtagtctta ctctcagttt 11880
tgcaacaact cagagtagaa tcatcatcta aattgtgggc tcaatgtgtc cagttacaca 11940
atgacattct cttagctaaa gatactactg aagcctttga aaaaatggtt tcactacttt 12000
ctgttttgct ttccatgcag ggtgctgtag acataaacaa gctttgtgaa gaaatgctgg 12060
acaacagggc aaccttacaa gctatagcct cagagtttag tttccttcca tcatatgcag 12120
cttttgctac tgctcaagaa gcttatgagc aggctgttgc taatggtgat tctgaagttg 12180
ttcttaaaaa gttgaagaag tctttgaatg tggctaaatc tgaatttgac cgtgatgcag 12240
ccatgcaacg taagttggaa aagatggctg atcaagctat gacccaaatg tataaacagg 12300
ctagatctga ggacaagagg gcaaaagtta ctagtgctat gcagacaatg cttttcacta 12360
tgcttagaaa gttggataat gatgcactca acaacattat caacaatgca agagatggtt 12420
gtgttccctt gaacataata cctcttacaa cagcagccaa actaatggtt gtcataccag 12480
actataacac atataaaaat acgtgtgatg gtacaacatt tacttatgca tcagcattgt 12540
gggaaatcca acaggttgta gatgcagata gtaaaattgt tcaacttagt gaaattagta 12600
tggacaattc acctaattta gcatggcctc ttattgtaac agctttaagg gccaattctg 12660
ctgtcaaatt acagaataat gagcttagtc ctgttgcact acgacagatg tcttgtgctg 12720
ccggtactac acaaactgct tgcactgatg acaatgcgtt agcttactac aacacaacaa 12780
ggggaggtaa gtttgtactt gcactgttat ccgatttaca ggatttgaaa tgggctagat 12840
tccctaagag tgatggaact ggtactatct atacagaact ggaaccacct tgtaggtttg 12900
ttacagacac acctaaaggt cctaaagtga agtatttata ctttattaaa ggattaaaca 12960
acctaaatag aggtatggta cttggtagtt tagctgccac agtacgtcta caagctggta 13020
atgcaacaga agtgcctgcc aattcaactg tattatcttt ctgtgctttt gctgtagatg 13080
ctgctaaagc ttacaaagat tatctagcta gtgggggaca accaatcact aattgtgtta 13140
agatgttgtg tacacacact ggtactggtc aggcaataac agttacaccg gaagccaata 13200
tggatcaaga atcctttggt ggtgcatcgt gttgtctgta ctgccgttgc cacatagatc 13260
atccaaatcc taaaggattt tgtgacttaa aaggtaagta tgtacaaata cctacaactt 13320
gtgctaatga ccctgtgggt tttacactta aaaacacagt ctgtaccgtc tgcggtatgt 13380
ggaaaggtta tggctgtagt tgtgatcaac tccgcgaacc catgcttcag tcagctgatg 13440
cacaatcgtt tttaaacggg tttgcggtgt aagtgcagcc cgtcttacac cgtgcggcac 13500
aggcactagt actgatgtcg tatacagggc ttttgacatc tacaatgata aagtagctgg 13560
ttttgctaaa ttcctaaaaa ctaattgttg tcgcttccaa gaaaaggacg aagatgacaa 13620
tttaattgat tcttactttg tagttaagag acatactttc tctaactacc aacatgaaga 13680
aacaatttat aatttactta aggattgtcc agctgttgct aaacatgact tctttaagtt 13740
tagaatagac ggtgacatgg taccacatat atcacgtcaa cgtcttacta aatacacaat 13800
ggcagacctc gtctatgctt taaggcattt tgatgaaggt aattgtgaca cattaaaaga 13860
aatacttgtc acatacaatt gttgtgatga tgattatttc aataaaaagg actggtatga 13920
ttttgtagaa aacccagata tattacgcgt atacgccaac ttaggtgaac gtgtacgcca 13980
agctttgtta aaaacagtac aattctgtga tgccatgcga aatgctggta ttgttggtgt 14040
actgacatta gataatcaag atctcaatgg taactggtat gatttcggtg atttcataca 14100
aaccacgcca ggtagtggag ttcctgttgt agattcttat tattcattgt taatgcctat 14160
attaaccttg accagggctt taactgcaga gtcacatgtt gacactgact taacaaagcc 14220
ttacattaag tgggatttgt taaaatatga cttcacggaa gagaggttaa aactctttga 14280
ccgttatttt aaatattggg atcagacata ccacccaaat tgtgttaact gtttggatga 14340
cagatgcatt ctgcattgtg caaactttaa tgttttattc tctacagtgt tcccacctac 14400
aagttttgga ccactagtga gaaaaatatt tgttgatggt gttccatttg tagtttcaac 14460
tggataccac ttcagagagc taggtgttgt acataatcag gatgtaaact tacatagctc 14520
tagacttagt tttaaggaat tacttgtgta tgctgctgac cctgctatgc acgctgcttc 14580
tggtaatcta ttactagata aacgcactac gtgcttttca gtagctgcac ttactaacaa 14640
tgttgctttt caaactgtca aacccggtaa ttttaacaaa gacttctatg actttgctgt 14700
gtctaagggt ttctttaagg aaggaagttc tgttgaatta aaacacttct tctttgctca 14760
ggatggtaat gctgctatca gcgattatga ctactatcgt tataatctac caacaatgtg 14820
tgatatcaga caactactat ttgtagttga agttgttgat aagtactttg attgttacga 14880
tggtggctgt attaatgcta accaagtcat cgtcaacaac ctagacaaat cagctggttt 14940
tccatttaat aaatggggta aggctagact ttattatgat tcaatgagtt atgaggatca 15000
agatgcactt ttcgcatata caaaacgtaa tgtcatccct actataactc aaatgaatct 15060
taagtatgcc attagtgcaa agaatagagc tcgcaccgta gctggtgtct ctatctgtag 15120
tactatgacc aatagacagt ttcatcaaaa attattgaaa tcaatagccg ccactagagg 15180
agctactgta gtaattggaa caagcaaatt ctatggtggt tggcacaaca tgttaaaaac 15240
tgtttatagt gatgtagaaa accctcacct tatgggttgg gattatccta aatgtgatag 15300
agccatgcct aacatgctta gaattatggc ctcacttgtt cttgctcgca aacatacaac 15360
gtgttgtagc ttgtcacacc gtttctatag attagctaat gagtgtgctc aagtattgag 15420
tgaaatggtc atgtgtggcg gttcactata tgttaaacca ggtggaacct catcaggaga 15480
tgccacaact gcttatgcta atagtgtttt taacatttgt caagctgtca cggccaatgt 15540
taatgcactt ttatctactg atggtaacaa aattgccgat aagtatgtcc gcaatttaca 15600
acacagactt tatgagtgtc tctatagaaa tagagatgtt gacacagact ttgtgaatga 15660
gttttacgca tatttgcgta aacatttctc aatgatgata ctctctgacg atgctgttgt 15720
gtgtttcaat agcacttatg catctcaagg tctagtggct agcataaaga actttaagtc 15780
agttctttat tatcaaaaca atgtttttat gtctgaagca aaatgttgga ctgagactga 15840
ccttactaaa ggacctcatg aattttgctc tcaacataca atgctagtta aacagggtga 15900
tgattatgtg taccttcctt acccagatcc atcaagaatc ctaggggccg gctgttttgt 15960
agatgatatc gtaaaaacag atggtacact tatgattgaa cggttcgtgt ctttagctat 16020
agatgcttac ccacttacta aacatcctaa tcaggagtat gctgatgtct ttcatttgta 16080
cttacaatac ataagaaagc tacatgatga gttaacagga cacatgttag acatgtattc 16140
tgttatgctt actaatgata acacttcaag gtattgggaa cctgagtttt atgaggctat 16200
gtacacaccg catacagtct tacaggctgt tggggcttgt gttctttgca attcacagac 16260
ttcattaaga tgtggtgctt gcatacgtag accattctta tgttgtaaat gctgttacga 16320
ccatgtcata tcaacatcac ataaattagt cttgtctgtt aatccgtatg tttgcaatgc 16380
tccaggttgt gatgtcacag atgtgactca actttactta ggaggtatga gctattattg 16440
taaatcacat aaaccaccca ttagttttcc attgtgtgct aatggacaag tttttggttt 16500
atataaaaat acatgtgttg gtagcgataa tgttactgac tttaatgcaa ttgcaacatg 16560
tgactggaca aatgctggtg attacatttt agctaacacc tgtactgaaa gactcaagct 16620
ttttgcagca gaaacgctca aagctactga ggagacattt aaactgtctt atggtattgc 16680
tactgtacgt gaagtgctgt ctgacagaga attacatctt tcatgggaag ttggtaaacc 16740
tagaccacca cttaaccgaa attatgtctt tactggttat cgtgtaacta aaaacagtaa 16800
agtacaaata ggagagtaca cctttgaaaa aggtgactat ggtgatgctg ttgtttaccg 16860
aggtacaaca acttacaaat taaatgttgg tgattatttt gtgctgacat cacatacagt 16920
aatgccatta agtgcaccta cactagtgcc acaagagcac tatgttagaa ttactggctt 16980
atacccaaca ctcaatatct cagatgagtt ttctagcaat gttgcaaatt atcaaaaggt 17040
tggtatgcaa aagtattcta cactccaggg accacctggt actggtaaga gtcattttgc 17100
tattggccta gctctctact acccttctgc tcgcatagtg tatacagctt gctctcatgc 17160
cgctgttgat gcactatgtg agaaggcatt aaaatatttg cctatagata aatgtagtag 17220
aattatacct gcacgtgctc gtgtagagtg ttttgataaa ttcaaagtga attcaacatt 17280
agaacagtat gtcttttgta ctgtaaatgc attgcctgag acgacagcag atatagttgt 17340
ctttgatgaa atttcaatgg ccacaaatta tgatttgagt gttgtcaatg ccagattacg 17400
tgctaagcac tatgtgtaca ttggcgaccc tgctcaatta cctgcaccac gcacattgct 17460
aactaagggc acactagaac cagaatattt caattcagtg tgtagactta tgaaaactat 17520
aggtccagac atgttcctcg gaacttgtcg gcgttgtcct gctgaaattg ttgacactgt 17580
gagtgctttg gtttatgata ataagcttaa agcacataaa gacaaatcag ctcaatgctt 17640
taaaatgttt tataagggtg ttatcacgca tgatgtttca tctgcaatta acaggccaca 17700
aataggcgtg gtaagagaat tccttacacg taaccctgct tggagaaaag ctgtctttat 17760
ttcaccttat aattcacaga atgctgtagc ctcaaagatt ttgggactac caattcaaac 17820
tgttgattca tcacagggct cagaatatga ctatgtcata ttcactcaaa ccactgaaac 17880
agctcactct tgtaatgtaa acagatttaa tgttgctatt accagagcaa aagtaggcat 17940
actttgcata atgtctgata gagaccttta tgacaagttg caatttacaa gtcttgaaat 18000
tccacgtagg aatgtggcaa ctttacaagc tgaaaatgta acaggactct ttaaagattg 18060
tagtaaggta atcactgggt tacatcctac acaggcacct acacacctca gtgttgacac 18120
taaattcaaa actgaaggtt tatgtgttga catacctggc atacctaagg acatgaccta 18180
tagaagactc atctctatga tgggttttaa aatgaattat caagttaatg gttaccctaa 18240
catgtttatc acccgcgaag aagctataag acatgtacgt gcatggattg gcttcgatgt 18300
cgaggggtgt catgctacta gagaagctgt tggtaccaat ttacctttac agctaggttt 18360
ttctacaggt gttaacctag ttgctgtacc tacaggttat gttgatacac ctaataatac 18420
agatttttcc agagttagtg ctaaaccacc gcctggagat caatttaaac acctcatacc 18480
acttatgtac aaaggacttc cttggaatgt agtgcgtata aagattgtac aaatgttaag 18540
tgacacactt aaaaatctct ctgacagagt cgtatttgtc ttatgggcac atggctttga 18600
gttgacatct atgaagtatt ttgtgaaaat aggacctgag cgcacctgtt gtctatgtga 18660
tagacgtgcc acatgctttt ccactgcttc agacacttat gcctgttggc atcattctat 18720
tggatttgat tacgtctata atccgtttat gattgatgtt caacaatggg gttttacagg 18780
taacctacaa agcaaccatg atctgtattg tcaagtccat ggtaatgcac atgtagctag 18840
ttgtgatgca atcatgacta ggtgtctagc tgtccacgag tgctttgtta agcgtgttga 18900
ctggactatt gaatatccta taattggtga tgaactgaag attaatgcgg cttgtagaaa 18960
ggttcaacac atggttgtta aagctgcatt attagcagac aaattcccag ttcttcacga 19020
cattggtaac cctaaagcta ttaagtgtgt acctcaagct gatgtagaat ggaagttcta 19080
tgatgcacag ccttgtagtg acaaagctta taaaatagaa gaattattct attcttatgc 19140
cacacattct gacaaattca cagatggtgt atgcctattt tggaattgca atgtcgatag 19200
atatcctgct aattccattg tttgtagatt tgacactaga gtgctatcta accttaactt 19260
gcctggttgt gatggtggca gtttgtatgt aaataaacat gcattccaca caccagcttt 19320
tgataaaagt gcttttgtta atttaaaaca attaccattt ttctattact ctgacagtcc 19380
atgtgagtct catggaaaac aagtagtgtc agatatagat tatgtaccac taaagtctgc 19440
tacgtgtata acacgttgca atttaggtgg tgctgtctgt agacatcatg ctaatgagta 19500
cagattgtat ctcgatgctt ataacatgat gatctcagct ggctttagct tgtgggttta 19560
caaacaattt gatacttata acctctggaa cacttttaca agacttcaga gtttagaaaa 19620
tgtggctttt aatgttgtaa ataagggaca ctttgatgga caacagggtg aagtaccagt 19680
ttctatcatt aataacactg tttacacaaa agttgatggt gttgatgtag aattgtttga 19740
aaataaaaca acattacctg ttaatgtagc atttgagctt tgggctaagc gcaacattaa 19800
accagtacca gaggtgaaaa tactcaataa tttgggtgtg gacattgctg ctaatactgt 19860
gatctgggac tacaaaagag atgctccagc acatatatct actattggtg tttgttctat 19920
gactgacata gccaagaaac caactgaaac gatttgtgca ccactcactg tcttttttga 19980
tggtagagtt gatggtcaag tagacttatt tagaaatgcc cgtaatggtg ttcttattac 20040
agaaggtagt gttaaaggtt tacaaccatc tgtaggtccc aaacaagcta gtcttaatgg 20100
agtcacatta attggagaag ccgtaaaaac acagttcaat tattataaga aagttgatgg 20160
tgttgtccaa caattacctg aaacttactt tactcagagt agaaatttac aagaatttaa 20220
acccaggagt caaatggaaa ttgatttctt agaattagct atggatgaat tcattgaacg 20280
gtataaatta gaaggctatg ccttcgaaca tatcgtttat ggagatttta gtcatagtca 20340
gttaggtggt ttacatctac tgattggact agctaaacgt tttaaggaat caccttttga 20400
attagaagat tttattccta tggacagtac agttaaaaac tatttcataa cagatgcgca 20460
aacaggttca tctaagtgtg tgtgttctgt tattgattta ttacttgatg attttgttga 20520
aataataaaa tcccaagatt tatctgtagt ttctaaggtt gtcaaagtga ctattgacta 20580
tacagaaatt tcatttatgc tttggtgtaa agatggccat gtagaaacat tttacccaaa 20640
attacaatct agtcaagcgt ggcaaccggg tgttgctatg cctaatcttt acaaaatgca 20700
aagaatgcta ttagaaaagt gtgaccttca aaattatggt gatagtgcaa cattacctaa 20760
aggcataatg atgaatgtcg caaaatatac tcaactgtgt caatatttaa acacattaac 20820
attagctgta ccctataata tgagagttat acattttggt gctggttctg ataaaggagt 20880
tgcaccaggt acagctgttt taagacagtg gttgcctacg ggtacgctgc ttgtcgattc 20940
agatcttaat gactttgtct ctgatgcaga ttcaactttg attggtgatt gtgcaactgt 21000
acatacagct aataaatggg atctcattat tagtgatatg tacgacccta agactaaaaa 21060
tgttacaaaa gaaaatgact ctaaagaggg ttttttcact tacatttgtg ggtttataca 21120
acaaaagcta gctcttggag gttccgtggc tataaagata acagaacatt cttggaatgc 21180
tgatctttat aagctcatgg gacacttcgc atggtggaca gcctttgtta ctaatgtgaa 21240
tgcgtcatca tctgaagcat ttttaattgg atgtaattat cttggcaaac cacgcgaaca 21300
aatagatggt tatgtcatgc atgcaaatta catattttgg aggaatacaa atccaattca 21360
gttgtcttcc tattctttat ttgacatgag taaatttccc cttaaattaa ggggtactgc 21420
tgttatgtct ttaaaagaag gtcaaatcaa tgatatgatt ttatctcttc ttagtaaagg 21480
tagacttata attagagaaa acaacagagt tgttatttct agtgatgttc ttgttaacaa 21540
ctaaacgaac aatgtttgtt tttcttgttt tattgccact agtctctagt cagtgtgtta 21600
atcttacaac cagaactcaa ttaccccctg catacactaa ttctttcaca cgtggtgttt 21660
attaccctga caaagttttc agatcctcag ttttacattc aactcaggac ttgttcttac 21720
ctttcttttc caatgttact tggttccatg ctatacatgt ctctgggacc aatggtacta 21780
agaggtttga taaccctgtc ctaccattta atgatggtgt ttattttgct tccattgaga 21840
agtctaacat aataagaggc tggatttttg gtactacttt agattcgaag acccagtccc 21900
tacttattgt taataacgct actaatgttg ttattaaagt ctgtgaattt caattttgta 21960
atgatccatt tttgggtgtt tattaccaca aaaacaacaa aagttggatg gaaagtgagt 22020
tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag ccttttctta 22080
tggaccttga aggaaaacag ggtaatttca aaaatcttag ggaatttgtg tttaagaata 22140
ttgatggtta ttttaaaata tattctaagc acacgcctat taatttagtg cgtgatctcc 22200
ctcagggttt ttcggcttta gaaccattgg tagatttgcc aataggtatt aacatcacta 22260
ggtttcaaac tttacttgct ttacatagaa gttatttgac tcctggtgat tcttcttcag 22320
gttggacagc tggtgctgca gcttattatg tgggttatct tcaacctagg acttttctat 22380
taaaatataa tgaaaatgga accattacag atgctgtaga ctgtgcactt gaccctctct 22440
cagaaacaaa gtgtacgttg aaatccttca ctgtagaaaa aggaatctat caaacttcta 22500
actttagagt ccaaccaaca gaatctattg ttagatttcc taatattaca aacttgtgcc 22560
cttttggtga agtttttaac gccaccagat ttgcatctgt ttatgcttgg aacaggaaga 22620
gaatcagcaa ctgtgttgct gattattctg tcctatataa ttccgcatca ttttccactt 22680
ttaagtgtta tggagtgtct cctactaaat taaatgatct ctgctttact aatgtctatg 22740
cagattcatt tgtaattaga ggtgatgaag tcagacaaat cgctccaggg caaactggaa 22800
agattgctga ttataattat aaattaccag atgattttac aggctgcgtt atagcttgga 22860
attctaacaa tcttgattct aaggttggtg gtaattataa ttacctgtat agattgttta 22920
ggaagtctaa tctcaaacct tttgagagag atatttcaac tgaaatctat caggccggta 22980
gcacaccttg taatggtgtt gaaggtttta attgttactt tcctttacaa tcatatggtt 23040
tccaacccac taatggtgtt ggttaccaac catacagagt agtagtactt tcttttgaac 23100
ttctacatgc accagcaact gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca 23160
aatgtgtcaa tttcaacttc aatggtttaa caggcacagg tgttcttact gagtctaaca 23220
aaaagtttct gcctttccaa caatttggca gagacattgc tgacactact gatgctgtcc 23280
gtgatccaca gacacttgag attcttgaca ttacaccatg ttcttttggt ggtgtcagtg 23340
ttataacacc aggaacaaat acttctaacc aggttgctgt tctttatcag gatgttaact 23400
gcacagaagt ccctgttgct attcatgcag atcaacttac tcctacttgg cgtgtttatt 23460
ctacaggttc taatgttttt caaacacgtg caggctgttt aataggggct gaacatgtca 23520
acaactcata tgagtgtgac atacccattg gtgcaggtat atgcgctagt tatcagactc 23580
agactaattc tcctcggcgg gcacgtagtg tagctagtca atccatcatt gcctacacta 23640
tgtcacttgg tgcagaaaat tcagttgctt actctaataa ctctattgcc atacccacaa 23700
attttactat tagtgttacc acagaaattc taccagtgtc tatgaccaag acatcagtag 23760
attgtacaat gtacatttgt ggtgattcaa ctgaatgcag caatcttttg ttgcaatatg 23820
gcagtttttg tacacaatta aaccgtgctt taactggaat agctgttgaa caagacaaaa 23880
acacccaaga agtttttgca caagtcaaac aaatttacaa aacaccacca attaaagatt 23940
ttggtggttt taatttttca caaatattac cagatccatc aaaaccaagc aagaggtcat 24000
ttattgaaga tctacttttc aacaaagtga cacttgcaga tgctggcttc atcaaacaat 24060
atggtgattg ccttggtgat attgctgcta gagacctcat ttgtgcacaa aagtttaacg 24120
gccttactgt tttgccacct ttgctcacag atgaaatgat tgctcaatac acttctgcac 24180
tgttagcggg tacaatcact tctggttgga cctttggtgc aggtgctgca ttacaaatac 24240
catttgctat gcaaatggct tataggttta atggtattgg agttacacag aatgttctct 24300
atgagaacca aaaattgatt gccaaccaat ttaatagtgc tattggcaaa attcaagact 24360
cactttcttc cacagcaagt gcacttggaa aacttcaaga tgtggtcaac caaaatgcac 24420
aagctttaaa cacgcttgtt aaacaactta gctccaattt tggtgcaatt tcaagtgttt 24480
taaatgatat cctttcacgt cttgacaaag ttgaggctga agtgcaaatt gataggttga 24540
tcacaggcag acttcaaagt ttgcagacat atgtgactca acaattaatt agagctgcag 24600
aaatcagagc ttctgctaat cttgctgcta ctaaaatgtc agagtgtgta cttggacaat 24660
caaaaagagt tgatttttgt ggaaagggct atcatcttat gtccttccct cagtcagcac 24720
ctcatggtgt agtcttcttg catgtgactt atgtccctgc acaagaaaag aacttcacaa 24780
ctgctcctgc catttgtcat gatggaaaag cacactttcc tcgtgaaggt gtctttgttt 24840
caaatggcac acactggttt gtaacacaaa ggaattttta tgaaccacaa atcattacta 24900
cagacaacac atttgtgtct ggtaactgtg atgttgtaat aggaattgtc aacaacacag 24960
tttatgatcc tttgcaacct gaattagact cattcaagga ggagttagat aaatatttta 25020
agaatcatac atcaccagat gttgatttag gtgacatctc tggcattaat gcttcagttg 25080
taaacattca aaaagaaatt gaccgcctca atgaggttgc caagaattta aatgaatctc 25140
tcatcgatct ccaagaactt ggaaagtatg agcagtatat aaaatggcca tggtacattt 25200
ggctaggttt tatagctggc ttgattgcca tagtaatggt gacaattatg ctttgctgta 25260
tgaccagttg ctgtagttgt ctcaagggct gttgttcttg tggatcctgc tgcaaatttg 25320
atgaagacga ctctgagcca gtgctcaaag gagtcaaatt acattacaca taaacgaact 25380
tatggatttg tttatgagaa tcttcacaat tggaactgta actttgaagc aaggtgaaat 25440
caaggatgct actccttcag attttgttcg cgctactgca acgataccga tacaagcctc 25500
actccctttc ggatggctta ttgttggcgt tgcacttctt gctgtttttc agagcgcttc 25560
caaaatcata accctcaaaa agcgatggca actagcactc tccaagggtg ttcactttgt 25620
ttgcaacttg ctgttgttgt ttgtaacagt ttactcacac cttttgctcg ttgctgctgg 25680
ccttgaagcc ccttttctct atctttatgc tttagtctac ttcttgcaga gtataaactt 25740
tgtaagaata ataatgaggc tttggctttg ctggaaatgc cgttccaaaa acccattact 25800
ttatgatgcc aactattttc tttgctggca tactaattgt tacgactatt gtatacctta 25860
caatagtgta acttcttcaa ttgtcattac ttcaggtgat ggcacaacaa gtcctatttc 25920
tgaacatgac taccagattg gtggttatac tgaaaaatgg gaatctggag taaaagactg 25980
tgttgtatta cacagttact tcacttcaga ctattaccag ctgtactcaa ctcaattgag 26040
tacagacact ggtgttgaac atgttacctt cttcatctac aataaaattg ttgatgagcc 26100
tgaagaacat gtccaaactc acacaatcga cggttcatcc ggagttgtta atccagtaat 26160
ggaaccaatt tatgatgaac cgacgacgac tactagcgtg cctttgtaag cacaagctga 26220
tgagtacgaa cttatgtact cattcgtttt ggaagagaca ggtacgttaa tagttaatag 26280
cgtacttctt tttcttgctt tcgtggtatt cttgctagtt acactagcca tccttactgc 26340
gcttcgattg tgtgcgtact gctgcaatat tgttaacgtg agtcttgtaa aaccttcttt 26400
ttacgtttac tctcgtgtta aaaatctgaa ttcttctaga gttcctgatc ttctggtcta 26460
aacgaactaa atattatatt agtttttctg tttggaactt taattttagc catggcagat 26520
tccagcggta ctattaccgt tgaagagctt aaaaagctcc ttgaacaatg gaacctagta 26580
ataggtttcc tattccttac atggatttgt cttctacaat ttgcctatgc caacaggaat 26640
aggtttttgt atataattaa gttaattttc ctctggctgt tatggccagt aactttagct 26700
tgttttgtgc ttgctgctgt ttacagaata aattggatca ccggtggaat tgctatcgca 26760
atggcttgtc ttgtaggctt gatgtggctc agctacttca ttgcttcttt cagactgttt 26820
gcgcgtacgc gttccatgtg gtcattcaat ccagaaacta acattcttct caacgtgcca 26880
ctccatggca ctattctgac cagaccgctt ctagaaagtg aactcgtaat cggagctgtg 26940
atccttcgtg gacatcttcg tattgctgga caccatctag gacgctgtga catcaaggac 27000
ctgcctaaag aaatcactgt tgctacatca cgaacgcttt cttattacaa attgggagct 27060
tcgcagcgtg tagcaggtga ctcaggtttt gctgcataca gtcgctacag gattggcaac 27120
tataaattaa acacagacca ttccagtagc agtgacaata ttgctttgct tgtacagtaa 27180
gtgacaacag atgtttcatc tcgttgactt tcaggttact atagcagaga tattactaat 27240
tattatgagg acttttaaag tttccatttg gaatcttgat tacatcataa acctcataat 27300
taaaaattta tctaagtcac taactgagaa taaatattct caattagatg aagagcaacc 27360
aatggagatt gattaaacga acatgaaaat tattcttttc ttggcactga taacactcgc 27420
tacttgtgag ctttatcact accaagagtg tgttagaggt acaacagtac ttttaaaaga 27480
accttgctct tctggaacat acgagggcaa ttcaccattt catcctctag ctgataacaa 27540
atttgcactg acttgcttta gcactcaatt tgcttttgct tgtcctgacg gcgtaaaaca 27600
cgtctatcag ttacgtgcca gatcagtttc acctaaactg ttcatcagac aagaggaagt 27660
tcaagaactt tactctccaa tttttcttat tgttgcggca atagtgttta taacactttg 27720
cttcacactc aaaagaaaga cagaatgatt gaactttcat taattgactt ctatttgtgc 27780
tttttagcct ttctgctatt ccttgtttta attatgctta ttatcttttg gttctcactt 27840
gaactgcaag atcataatga aacttgtcac gcctaaacga acatgaaatt tcttgttttc 27900
ttaggaatca tcacaactgt agctgcattt caccaagaat gtagtttaca gtcatgtact 27960
caacatcaac catatgtagt tgatgacccg tgtcctattc acttctattc taaatggtat 28020
attagagtag gagctagaaa atcagcacct ttaattgaat tgtgcgtgga tgaggctggt 28080
tctaaatcac ccattcagta catcgatatc ggtaattata cagtttcctg tttacctttt 28140
acaattaatt gccaggaacc taaattgggt agtcttgtag tgcgttgttc gttctatgaa 28200
gactttttag agtatcatga cgttcgtgtt gttttagatt tcatctaaac gaacaaacta 28260
aaatgtctga taatggaccc caaaatcagc gaaatgcacc ccgcattacg tttggtggac 28320
cctcagattc aactggcagt aaccagaatg gagaacgcag tggggcgcga tcaaaacaac 28380
gtcggcccca aggtttaccc aataatactg cgtcttggtt caccgctctc actcaacatg 28440
gcaaggaaga ccttaaattc cctcgaggac aaggcgttcc aattaacacc aatagcagtc 28500
cagatgacca aattggctac taccgaagag ctaccagacg aattcgtggt ggtgacggta 28560
aaatgaaaga tctcagtcca agatggtatt tctactacct aggaactggg ccagaagctg 28620
gacttcccta tggtgctaac aaagacggca tcatatgggt tgcaactgag ggagccttga 28680
atacaccaaa agatcacatt ggcacccgca atcctgctaa caatgctgca atcgtgctac 28740
aacttcctca aggaacaaca ttgccaaaag gcttctacgc agaagggagc agaggcggca 28800
gtcaagcctc ttctcgttcc tcatcacgta gtcgcaacag ttcaagaaat tcaactccag 28860
gcagcagtag gggaacttct cctgctagaa tggctggcaa tggcggtgat gctgctcttg 28920
ctttgctgct gcttgacaga ttgaaccagc ttgagagcaa aatgtctggt aaaggccaac 28980
aacaacaagg ccaaactgtc actaagaaat ctgctgctga ggcttctaag aagcctcggc 29040
aaaaacgtac tgccactaaa gcatacaatg taacacaagc tttcggcaga cgtggtccag 29100
aacaaaccca aggaaatttt ggggaccagg aactaatcag acaaggaact gattacaaac 29160
attggccgca aattgcacaa tttgccccca gcgcttcagc gttcttcgga atgtcgcgca 29220
ttggcatgga agtcacacct tcgggaacgt ggttgaccta cacaggtgcc atcaaattgg 29280
atgacaaaga tccaaatttc aaagatcaag tcattttgct gaataagcat attgacgcat 29340
acaaaacatt cccaccaaca gagcctaaaa aggacaaaaa gaagaaggct gatgaaactc 29400
aagccttacc gcagagacag aagaaacagc aaactgtgac tcttcttcct gctgcagatt 29460
tggatgattt ctccaaacaa ttgcaacaat ccatgagcag tgctgactca actcaggcct 29520
aaactcatgc agaccacaca aggcagatgg gctatataaa cattttcgct tttccgttta 29580
cgatatatag tctactcttg tgcagaatga attctcgtaa ctacatagca caagtagatg 29640
tagttaactt taatctcaca tagcaatctt taatcagtgt gtaacattag ggaggacttg 29700
aaagagccac cacattttca ccgaggccac gcggagtacg atcgagtgta cagtgaacaa 29760
tgctagggag agctgcctat atggaagagc cctaatgtgt aaaattaatt ttagtagtgc 29820
tatccccatg tgattttaat agcttcttar gaswmtswca aaaaaaaaaa aaaaaaaaaa 29880
<210> 2
<211> 29891
<212> DNA
<213> SARS-CoV-2(Artificial Sequence)
<400> 2
attaaaggtt tataccttcc caggtaacaa accaaccaac tttcgatctc ttgtagatct 60
gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc ttagtgcact 120
cacgcagtat aattaataac taattactgt cgttgacagg acacgagtaa ctcgtctatc 180
ttctgcaggc tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac atctaggttt 240
cgtccgggtg tgaccgaaag gtaagatgga gagccttgtc cctggtttca acgagaaaac 300
acacgtccaa ctcagtttgc ctgttttaca ggttcgcgac gtgctcgtac gtggctttgg 360
agactccgtg gaggaggtct tatcagaggc acgtcaacat cttaaagatg gcacttgtgg 420
cttagtagaa gttgaaaaag gcgttttgcc tcaacttgaa cagccctatg tgttcatcaa 480
acgttcggat gctcgaactg cacctcatgg tcatgttatg gttgagctgg tagcagaact 540
cgaaggcatt cagtacggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg 600
cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg 660
tggccatagt tacggcgccg atctaaagtc atttgactta ggcgacgagc ttggcactga 720
tccttatgaa gattttcaag aaaactggaa cactaaacat agcagtggtg ttacccgtga 780
actcatgcgt gagcttaacg gaggggcata cactcgctat gtcgataaca acttctgtgg 840
ccctgatggc taccctcttg agtgcattaa agaccttcta gcacgtgctg gtaaagcttc 900
atgcactttg tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg 960
tgaacatgag catgaaattg cttggtacac ggaacgttct gaaaagagct atgaattgca 1020
gacacctttt gaaattaaat tggcaaagaa atttgacacc ttcaatgggg aatgtccaaa 1080
ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg aaaagaaaaa 1140
gcttgatggc tttatgggta gaattcgatc tgtctatcca gttgcgtcac caaatgaatg 1200
caaccaaatg tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca 1260
gacgggcgat tttgttaaag ccacttgcga attttgtggc actgagaatt tgactaaaga 1320
aggtgccact acttgtggtt acttacccca aaatgctgtt gttaaaattt attgtccagc 1380
atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata atgaatctgg 1440
cttgaaaacc attcttcgta agggtggtcg cactattgcc tttggaggct gtgtgttctc 1500
ttatgttggt tgccataaca agtgtgccta ttgggttcca cgtgctagcg ctaacatagg 1560
ttgtaaccat acaggtgttg ttggagaagg ttccgaaggt cttaatgaca accttcttga 1620
aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac ttaatgaaga 1680
gatcgccatt attttggcat ctttttctgc ttccacaagt gcttttgtgg aaactgtgaa 1740
aggtttggat tataaagcat tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac 1800
aaaaggaaaa gctaaaaaag gtgcctggaa tattggtgaa cagaaatcaa tactgagtcc 1860
tctttatgca tttgcatcag aggctgctcg tgttgtacga tcaattttct cccgcactct 1920
tgaaactgct caaaattctg tgcgtgtttt acagaaggcc gctataacaa tactagatgg 1980
aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac 2040
taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg 2100
gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga 2160
agagaagttt aaggaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat 2220
ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa 2280
ggagagtgtt cagacattct ttaagcttgt aaataaattt ttggctttgt gtgctgactc 2340
tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca 2400
ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgcc 2460
tctaaaagcc ccaaaagaaa ttatcttctt agagggagaa acacttccca cagaagtgtt 2520
aacagaggaa gttgtcttga aaactggtga tttacaacca ttagaacaac ctactagtga 2580
agctgttgaa gctccattgg ttggtacacc agtttgtatt aacgggctta tgttgctcga 2640
aatcaaagac acagaaaagt actgtgccct tgcacctaat atgatggtaa caaacaatac 2700
cttcacactc aaaggcggtg caccaacaaa ggttactttt ggtgatgaca ctgtgataga 2760
agtgcaaggt tacaagagtg tgaatatcac ttttgaactt gatgaaagga ttgataaagt 2820
acttaatgag aagtgctctg cctatacagt tgaactcggt acagaagtaa atgagttcgc 2880
ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacacc 2940
actgggcatt gatttagatg agtggagtat ggctacatac tacttatttg atgagtctgg 3000
tgagtttaaa ttggcttcac atatgtattg ttctttctac cctccagatg aggatgaaga 3060
agaaggtgat tgtgaagaag aagagtttga gccatcaact caatatgagt atggtactga 3120
agatgattac caaggtaaac ctttggaatt tggtgccact tctgctgctc ttcaacctga 3180
agaagagcaa gaagaagatt ggttagatga tgatagtcaa caaactgttg gtcaacaaga 3240
cggcagtgag gacaatcaga caactactat tcaaacaatt gttgaggttc aacctcaatt 3300
agagatggaa cttacaccag ttgttcagac tattgaagtg aatagtttta gtggttattt 3360
aaaacttact gacaatgtat acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt 3420
aaaaccaaca gtggttgtta atgcagccaa tgtttacctt aaacatggag gaggtgttgc 3480
aggagcctta aataaggcta ctaacaatgc catgcaagtt gaatctgatg attacatagc 3540
tactaatgga ccacttaaag tgggtggtag ttgtgtttta agcggacaca atcttgctaa 3600
acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacattc aacttcttaa 3660
gagtgcttat gaaaatttta atcagcacga agttctactt gcaccattat tatcagctgg 3720
tatttttggt gctgacccta tacattcttt aagagtttgt gtagatactg ttcgcacaaa 3780
tgtctactta gctgtctttg ataaaaatct ctatgacaaa cttgtttcaa gctttttgga 3840
aatgaagagt gaaaagcaag ttgaacaaaa gatcgctgag attcctaaag aggaagttaa 3900
gccatttata actgaaagta aaccttcagt tgaacagaga aaacaagatg ataagaaaat 3960
caaagcttgt gttgaagaag ttacaacaac tctggaagaa actaagttcc tcacagaaaa 4020
cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca ctcttgttag 4080
tgacattgac atcactttct taaagaaaga tgctccatat atagtgggtg atgttgttca 4140
agagggtgtt ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat 4200
gctagcgaaa gctttgagaa aagtgccaac agacaattat ataaccactt acccgggtca 4260
gggtttaaat ggttacactg tagaggaggc aaagacagtg cttaaaaagt gtaaaagtgc 4320
cttttacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg gaactgtttc 4380
ttggaatttg cgagaaatgc ttgcacatgc agaagaaaca cgcaaattaa tgcctgtctg 4440
tgtggaaact aaagccatag tttcaactat acagcgtaaa tataagggta ttaaaataca 4500
agagggtgtg gttgattatg gtgctagatt ttacttttac accagtaaaa caactgtagc 4560
gtcacttatc aacacactta acgatctaaa tgaaactctt gttacaatgc cacttggcta 4620
tgtaacacat ggcttaaatt tggaagaagc tgctcggtat atgagatctc tcaaagtgcc 4680
agctacagtt tctgtttctt cacctgatgc tgttacagcg tataatggtt atcttacttc 4740
ttcttctaaa acacctgaag aacattttat tgaaaccatc tcacttgctg gttcctataa 4800
agattggtcc tattctggac aatctacaca actaggtata gaatttctta agagaggtga 4860
taaaagtgta tattacacta gtaatcctac cacattccac ctagatggtg aagttatcac 4920
ctttgacaat cttaagacac ttctttcttt gagagaagtg aggactatta aggtgtttac 4980
aacagtagac aacattaacc tccacacgca agttgtggac atgtcaatga catatggaca 5040
acagtttggt ccaacttatt tggatggagc tgatgttact aaaataaaac ctcataattc 5100
acatgaaggt aaaacatttt atgttttacc taatgatgac actctacgtg ttgaggcttt 5160
tgagtactac cacacaactg atcctagttt tctgggtagg tacatgtcag cattaaatca 5220
cactaaaaag tggaaatacc cacaagttaa tggtttaact tctattaaat gggcagataa 5280
caactgttat cttgccactg cattgttaac actccaacaa atagagttga agtttaatcc 5340
acctgctcta caagatgctt attacagagc aagggctggt gaagctgcta acttttgtgc 5400
acttatctta gcctactgta ataagacagt aggtgagtta ggtgatgtta gagaaacaat 5460
gagttacttg tttcaacatg ccaatttaga ttcttgcaaa agagtcttga acgtggtgtg 5520
taaaacttgt ggacaacagc agacaaccct taagggtgta gaagctgtta tgtacatggg 5580
cacactttct tatgaacaat ttaagaaagg tgttcagata ccttgtacgt gtggtaaaca 5640
agctacaaaa tatctagtac aacaggagtc accttttgtt atgatgtcag caccacctgc 5700
tcagtatgaa cttaagcatg gtacatttac ttgtgctagt gagtacactg gtaattacca 5760
gtgtggtcac tataaacata taacttctaa agaaactttg tattgcatag acggtgcttt 5820
acttacaaag tcctcagaat acaaaggtcc tattacggat gttttctaca aagaaaacag 5880
ttacacaaca accataaaac cagttactta taaattggat ggtgttgttt gtacagaaat 5940
tgaccctaag ttggacaatt attataagaa agacaattct tatttcacag agcaaccaat 6000
tgatcttgta ccaaaccaac catatccaaa cgcaagcttc gataatttta agtttgtatg 6060
tgataatatc aaatttgctg atgatttaaa ccagttaact ggttataaga aacctgcttc 6120
aagagagctt aaagttacat ttttccctga cttaaatggt gatgtggtgg ctattgatta 6180
taaacactac acaccctctt ttaagaaagg agctaaattg ttacataaac ctattgtttg 6240
gcatgttaac aatgcaacta ataaagccac gtataaacca aatacctggt gtatacgttg 6300
tctttggagc acaaaaccag ttgaaacatc aaattcgttt gatgtactga agtcagagga 6360
cgcgcaggga atggataatc ttgcctgcga agatctaaaa ccagtctctg aagaagtagt 6420
ggaaaatcct accatacaga aagacgttct tgagtgtaat gtgaaaacta ccgaagttgt 6480
aggagacatt atacttaaac cagcaaataa tagtttaaaa attacagaag aggttggcca 6540
cacagatcta atggctgctt atgtagacaa ttctagtctt actattaaga aacctaatga 6600
attatctaga gtattaggtt tgaaaaccct tgctactcat ggtttagctg ctgttaatag 6660
tgtcccttgg gatactatag ctaattatgc taagcctttt cttaacaaag ttgttagtac 6720
aactactaac atagttacac ggtgtttaaa ccgtgtttgt actaattata tgccttattt 6780
ctttacttta ttgctacaat tgtgtacttt tactagaagt acaaattcta gaattaaagc 6840
atctatgccg actactatag caaagaatac tgttaagagt gtcggtaaat tttgtctaga 6900
ggcttcattt aattatttga agtcacctaa tttttctaaa ctgataaata ttataatttg 6960
gtttttacta ttaagtgttt gcctaggttc tttaatctac tcaaccgctg ctttaggtgt 7020
tttaatgtct aatttaggca tgccttctta ctgtactggt tacagagaag gctatttgaa 7080
ctctactaat gtcactattg caacctactg tactggttct ataccttgta gtgtttgtct 7140
tagtggttta gattctttag acacctatcc ttctttagaa actatacaaa ttaccatttc 7200
atcttttaaa tgggatttaa ctgcttttgg cttagttgca gagtggtttt tggcatatat 7260
tcttttcact aggtttttct atgtacttgg attggctgca atcatgcaat tgtttttcag 7320
ctattttgca gtacatttta ttagtaattc ttggcttatg tggttaataa ttaatcttgt 7380
acaaatggcc ccgatttcag ctatggttag aatgtacatc ttctttgcat cattttatta 7440
tgtatggaaa agttatgtgc atgttgtaga cggttgtaat tcatcaactt gtatgatgtg 7500
ttacaaacgt aatagagcaa caagagtcga atgtacaact attgttaatg gtgttagaag 7560
gtccttttat gtctatgcta atggaggtaa aggcttttgc aaactacaca attggaattg 7620
tgttaattgt gatacattct gtgctggtag tacatttatt agtgatgaag ttgcgagaga 7680
cttgtcacta cagtttaaaa gaccaataaa tcctactgac cagtcttctt acatcgttga 7740
tagtgttaca gtgaagaatg gttccatcca tctttacttt gataaagctg gtcaaaagac 7800
ttatgaaaga cattctctct ctcattttgt taacttagac aacctgagag ctaataacac 7860
taaaggttca ttgcctatta atgttatagt ttttgatggt aaatcaaaat gtgaagaatc 7920
atctgcaaaa tcagcgtctg tttactacag tcagcttatg tgtcaaccta tactgttact 7980
agatcaggca ttagtgtctg atgttggtga tagtgcggaa gttgcagtta aaatgtttga 8040
tgcttacgtt aatacgtttt catcaacttt taacgtacca atggaaaaac tcaaaacact 8100
agttgcaact gcagaagctg aacttgcaaa gaatgtgtcc ttagacaatg tcttatctac 8160
ttttatttca gcagctcggc aagggtttgt tgattcagat gtagaaacta aagatgttgt 8220
tgaatgtctt aaattgtcac atcaatctga catagaagtt actggcgata gttgtaataa 8280
ctatatgctc acctataaca aagttgaaaa catgacaccc cgtgaccttg gtgcttgtat 8340
tgactgtagt gcgcgtcata ttaatgcgca ggtagcaaaa agtcacaaca ttgctttgat 8400
atggaacgtt aaagatttca tgtcattgtc tgaacaacta cgaaaacaaa tacgtagtgc 8460
tgctaaaaag aataacttac cttttaagtt gacatgtgca actactagac aagttgttaa 8520
tgttgtaaca acaaagatag cacttaaggg tggtaaaatt gttaataatt ggttgaagca 8580
gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt taataacacc 8640
tgttcatgtc atgtctaaac atactgactt ttcaagtgaa atcataggat acaaggctat 8700
tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc 8760
tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc 8820
attgattgct gcagtcataa caagagaagt gggttttgtc gtgcctggtt tgcctggcac 8880
gatattacgc acaactaatg gtgacttttt gcatttctta cctagagttt ttagtgcagt 8940
tggtaacatc tgttacacac catcaaaact tatagagtac actgactttg caacatcagc 9000
ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaagc cagtaccata 9060
ttgttatgat accaatgtac tagaaggttc tgttgcttat gaaagtttac gccctgacac 9120
acgttatgtg ctcatggatg gctctattat tcaatttcct aacacctacc ttgaaggttc 9180
tgttagagtg gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagatc 9240
agaagctggt gtttgtgtat ctactagtgg tagatgggta cttaacaatg attattacag 9300
atctttacca ggagttttct gtggtgtaga tgctgtaaat ttacttacta atatgtttac 9360
accactaatt caacctattg gtgctttgga catatcagca tctatagtag ctggtggtat 9420
tgtagctatc gtagtaacat gccttgccta ctattttatg aggtttagaa gagcttttgg 9480
tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat tcactgtact 9540
ctgtttaaca ccagtttact cattcttacc tggtgtttat tctgttattt acttgtactt 9600
gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt 9660
cacaccttta gtacctttct ggataacaat tgcttatatc atttgtattt ccacaaagca 9720
tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9780
tagtactttt gaagaagctg cgctgtgcac ctttttgtta aataaagaaa tgtatctaaa 9840
gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa 9900
taagtacaag tattttagtg gagcaatgga tacaactagc tacagagaag ctgcttgttg 9960
tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgttc tttaccaacc 10020
accacaaacc tctatcacct cagctgtttt gcagagtggt tttagaaaaa tggcattccc 10080
atctggtaaa gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaacgg 10140
tctttggctt gatgacgtag tttactgtcc aagacatgtg atctgcacct ctgaagacat 10200
gcttaaccct aattatgaag atttactcat tcgtaagtct aatcataatt tcttggtaca 10260
ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg tacttaagct 10320
taaggttgat acagccaatc ctaagacacc taagtataag tttgttcgca ttcaaccagg 10380
acagactttt tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc 10440
tatgaggccc aatttcacta ttaagggttc attccttaat ggttcatgtg gtagtgttgg 10500
ttttaacata gattatgact gtgtctcttt ttgttacatg caccatatgg aattaccaac 10560
tggagttcat gctggcacag acttagaagg taacttttat ggaccttttg ttgacaggca 10620
aacagcacaa gcagctggta cggacacaac tattacagtt aatgttttag cttggttgta 10680
cgctgctgtt ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga 10740
ctttaacctt gtggctatga agtacaatta tgaacctcta acacaagacc atgttgacat 10800
actaggacct ctttctgctc aaactggaat tgccgtttta gatatgtgtg cttcattaaa 10860
agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt tattagaaga 10920
tgaatttaca ccttttgatg ttgttagaca atgctcaggt gttactttcc aaagtgcagt 10980
gaaaagaaca atcaagggta cacaccactg gttgttactc acaattttga cttcactttt 11040
agttttagtc cagagtactc aatggtcttt gttctttttt ttgtatgaaa atgccttttt 11100
accttttgct atgggtatta ttgctatgtc tgcttttgca atgatgtttg tcaaacataa 11160
gcatgcattt ctctgtttgt ttttgttacc ttctcttgcc actgtagctt attttaatat 11220
ggtctatatg cctgctagtt gggtgatgcg tattatgaca tggttggata tggttgatac 11280
tagtttgtct ggttttaagc taaaagactg tgttatgtat gcatcagctg tagtgttact 11340
aatccttatg acagcaagaa ctgtgtatga tgatggtgct aggagagtgt ggacacttat 11400
gaatgtcttg acactcgttt ataaagttta ttatggtaat gctttagatc aagccatttc 11460
catgtgggct cttataatct ctgttacttc taactactca ggtgtagtta caactgtcat 11520
gtttttggcc agaggtattg tttttatgtg tgttgagtat tgccctattt tcttcataac 11580
tggtaataca cttcagtgta taatgctagt ttattgtttc ttaggctatt tttgtacttg 11640
ttactttggc ctcttttgtt tactcaaccg ctactttaga ctgactcttg gtgtttatga 11700
ttacttagtt tctacacagg agtttagata tatgaattca cagggactac tcccacccaa 11760
gaatagcata gatgccttca aactcaacat taaattgttg ggtgttggtg gcaaaccttg 11820
tatcaaagta gccactgtac agtctaaaat gtcagatgta aagtgcacat cagtagtctt 11880
actctcagtt ttgcaacaac tcagagtaga atcatcatct aaattgtggg ctcaatgtgt 11940
ccagttacac aatgacattc tcttagctaa agatactact gaagcctttg aaaaaatggt 12000
ttcactactt tctgttttgc tttccatgca gggtgctgta gacataaaca agctttgtga 12060
agaaatgctg gacaacaggg caaccttaca agctatagcc tcagagttta gttcccttcc 12120
atcatatgca gcttttgcta ctgctcaaga agcttatgag caggctgttg ctaatggtga 12180
ttctgaagtt gttcttaaaa agttgaagaa gtctttgaat gtggctaaat ctgaatttga 12240
ccgtgatgca gccatgcaac gtaagttgga aaagatggct gatcaagcta tgacccaaat 12300
gtataaacag gctagatctg aggacaagag ggcaaaagtt actagtgcta tgcagacaat 12360
gcttttcact atgcttagaa agttggataa tgatgcactc aacaacatta tcaacaatgc 12420
aagagatggt tgtgttccct tgaacataat acctcttaca acagcagcca aactaatggt 12480
tgtcatacca gactataaca catataaaaa tacgtgtgat ggtacaacat ttacttatgc 12540
atcagcattg tgggaaatcc aacaggttgt agatgcagat agtaaaattg ttcaacttag 12600
tgaaattagt atggacaatt cacctaattt agcatggcct cttattgtaa cagctttaag 12660
ggccaattct gctgtcaaat tacagaataa tgagcttagt cctgttgcac tacgacagat 12720
gtcttgtgct gccggtacta cacaaactgc ttgcactgat gacaatgcgt tagcttacta 12780
caacacaaca aagggaggta ggtttgtact tgcactgtta tccgatttac aggatttgaa 12840
atgggctaga ttccctaaga gtgatggaac tggtactatc tatacagaac tggaaccacc 12900
ttgtaggttt gttacagaca cacctaaagg tcctaaagtg aagtatttat actttattaa 12960
aggattaaac aacctaaata gaggtatggt acttggtagt ttagctgcca cagtacgtct 13020
acaagctggt aatgcaacag aagtgcctgc caattcaact gtattatctt tctgtgcttt 13080
tgctgtagat gctgctaaag cttacaaaga ttatctagct agtgggggac aaccaatcac 13140
taattgtgtt aagatgttgt gtacacacac tggtactggt caggcaataa cagttacacc 13200
ggaagccaat atggatcaag aatcctttgg tggtgcatcg tgttgtctgt actgccgttg 13260
ccacatagat catccaaatc ctaaaggatt ttgtgactta aaaggtaagt atgtacaaat 13320
acctacaact tgtgctaatg accctgtggg ttttacactt aaaaacacag tctgtaccgt 13380
ctgcggtatg tggaaaggtt atggctgtag ttgtgatcaa ctccgcgaac ccatgcttca 13440
gtcagctgat gcacaatcgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca 13500
ccgtgcggca caggcactag tactgatgtc gtatacaggg cttttgacat ctacaatgat 13560
aaagtagctg gttttgctaa attcctaaaa actaattgtt gtcgcttcca agaaaaggac 13620
gaagatgaca atttaattga ttcttacttt gtagttaaga gacacacttt ctctaactac 13680
caacatgaag aaacaattta taatttactt aaggattgtc cagctgttgc taaacatgac 13740
ttctttaagt ttagaataga cggtgacatg gtaccacata tatcacgtca acgtcttact 13800
aaatacacaa tggcagacct cgtctatgct ttaaggcatt ttgatgaagg taattgtgac 13860
acattaaaag aaatacttgt cacatacaat tgttgtgatg atgattattt caataaaaag 13920
gactggtatg attttgtaga aaacccagat atattacgcg tatacgccaa cttaggtgaa 13980
cgtgtacgcc aagctttgtt aaaaacagta caattctgtg atgccatgcg aaatgctggt 14040
attgttggtg tactgacatt agataatcaa gatctcaatg gtaactggta tgatttcggt 14100
gatttcatac aaaccacgcc aggtagtgga gttcctgttg tagattctta ttattcattg 14160
ttaatgccta tattaacctt gaccagggct ttaactgcag agtcacatgt tgacactgac 14220
ttaacaaagc cttacattaa gtgggatttg ttaaaatatg acttcacgga agagaggtta 14280
aaactctttg accgttattt taaatattgg gatcagacat accacccaaa ttgtgttaac 14340
tgtttggatg acagatgcat tctgcattgt gcaaacttta atgttttatt ctctacagtg 14400
ttcccaccta caagttttgg accactagtg agaaaaatat ttgttgatgg tgttccattt 14460
gtagtttcaa ctggatacca cttcagagag ctaggtgttg tacataatca ggatgtaaac 14520
ttacatagct ctagacttag ttttaaggaa ttacttgtgt atgctgctga ccctgctatg 14580
cacgctgctt ctggtaatct attactagat aaacgcacta cgtgcttttc agtagctgca 14640
cttactaaca atgttgcttt tcaaactgtc aaacccggta attttaacaa agacttctat 14700
gactttgctg tgtctaaggg tttctttaag gaaggaagtt ctgttgaatt aaaacacttc 14760
ttctttgctc aggatggtaa tgctgctatc agcgattatg actactatcg ttataatcta 14820
ccaacaatgt gtgatatcag acaactacta tttgtagttg aagttgttga taagtacttt 14880
gattgttacg atggtggctg tattaatgct aaccaagtca tcgtcaacaa cctagacaaa 14940
tcagctggtt ttccatttaa taaatggggt aaggctagac tttattatga ttcaatgagt 15000
tatgaggatc aagatgcact tttcgcatat acaaaacgta atgtcatccc tactataact 15060
caaatgaatc ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc 15120
tctatctgta gtactatgac caatagacag tttcatcaaa aattattgaa atcaatagcc 15180
gccactagag gagctactgt agtaattgga acaagcaaat tctatggtgg ttggcacaac 15240
atgttaaaaa ctgtttatag tgatgtagaa aaccctcacc ttatgggttg ggattatcct 15300
aaatgtgata gagccatgcc taacatgctt agaattatgg cctcacttgt tcttgctcgc 15360
aaacatacaa cgtgttgtag cttgtcacac cgtttctata gattagctaa tgagtgtgct 15420
caagtattga gtgaaatggt catgtgtggc ggttcactat atgttaaacc aggtggaacc 15480
tcatcaggag atgccacaac tgcttatgct aatagtgttt ttaacatttg tcaagctgtc 15540
acggccaatg ttaatgcact tttatctact gatggtaaca aaattgccga taagtatgtc 15600
cgcaatttac aacacagact ttatgagtgt ctctatagaa atagagatgt tgacacagac 15660
tttgtgaatg agttttacgc atatttgcgt aaacatttct caatgatgat actctctgac 15720
gatgctgttg tgtgtttcaa tagcacttat gcatctcaag gtctagtggc tagcataaag 15780
aactttaagt cagttcttta ttatcaaaac aatgttttta tgtctgaagc aaaatgttgg 15840
actgagactg accttactaa aggacctcat gaattttgct ctcaacatac aatgctagtt 15900
aaacagggtg atgattatgt gtaccttcct tacccagatc catcaagaat cctaggggcc 15960
ggctgttttg tagatgatat cgtaaaaaca gatggtacac ttatgattga acggttcgtg 16020
tctttagcta tagatgctta cccacttact aaacatccta atcaggagta tgctgatgtc 16080
tttcatttgt acttacaata cataagaaag ctacatgatg agttaacagg acacatgtta 16140
gacatgtatt ctgttatgct tactaatgat aacacttcaa ggtattggga acctgagttt 16200
tatgaggcta tgtacacacc gcatacagtc ttacaggctg ttggggcttg tgttctttgc 16260
aattcacaga cttcattaag atgtggtgct tgcatacgta gaccattctt atgttgtaaa 16320
tgctgttacg accatgtcat atcaacatca cataaattag tcttgtctgt taatccgtat 16380
gtttgcaatg ctccaggttg tgatgtcaca gatgtgactc aactttactt aggaggtatg 16440
agctattatt gtaaatcaca taaaccaccc attagttttc cattgtgtgc taatggacaa 16500
gtttttggtt tatataaaaa tacatgtgtt ggtagcgata atgttactga ctttaatgca 16560
attgcaacat gtgactggac aaatgctggt gattacattt tagctaacac ctgtactgaa 16620
agactcaagc tttttgcagc agaaacgctc aaagctactg aggagacatt taaactgtct 16680
tatggtattg ctactgtacg tgaagtgctg tctgacagag aattacatct ttcatgggaa 16740
gttggtaaac ctagaccacc acttaaccga aattatgtct ttactggtta tcgtgtaact 16800
aaaaacagta aagtacaaat aggagagtac acctttgaaa aaggtgacta tggtgatgct 16860
gttgtttacc gaggtacaac aacttacaaa ttaaatgttg gtgattattt tgtgctgaca 16920
tcacatacag taatgccatt aagtgcacct acactagtgc cacaagagca ctatgttaga 16980
attactggct tatacccaac actcaatatc tcagatgagt tttctagcaa tgttgcaaat 17040
tatcaaaagg ttggtatgca aaagtattct acactccagg gaccacctgg tactggtaag 17100
agtcattttg ctattggcct agctctctac tacccttctg ctcgcatagt gtatacagct 17160
tgctctcatg ccgctgttga tgcactatgt gagaaggcat taaaatattt gcctatagat 17220
aaatgtagta gaattatacc tgcacgtgct cgtgtagagt gttttgataa attcaaagtg 17280
aattcaacat tagaacagta tgtcttttgt actgtaaatg cattgcctga gacgacagca 17340
gatatagttg tctttgatga aatttcaatg gccacaaatt atgatttgag tgttgtcaat 17400
gccagattac gtgctaagca ctatgtgtac attggcgacc ctgctcaatt acctgcacca 17460
cgcacattgc taactaaggg cacactagaa ccagaatatt tcaattcagt gtgtagactt 17520
atgaaaacta taggtccaga catgttcctc ggaacttgtc ggcgttgtcc tgctgaaatt 17580
gttgacactg tgagtgcttt ggtttatgat aataagctta aagcacataa agacaaatca 17640
gctcaatgct ttaaaatgtt ttataagggt gttatcacgc atgatgtttc atctgcaatt 17700
aacaggccac aaataggcgt ggtaagagaa ttccttacac gtaaccctgc ttggagaaaa 17760
gctgtcttta tttcacctta taattcacag aatgctgtag cctcaaagat tttgggacta 17820
ccaactcaaa ctgttgattc atcacagggc tcagaatatg actatgtcat attcactcaa 17880
accactgaaa cagctcactc ttgtaatgta aacagattta atgttgctat taccagagca 17940
aaagtaggca tactttgcat aatgtctgat agagaccttt atgacaagtt gcaatttaca 18000
agtcttgaaa ttccacgtag gaatgtggca actttacaag ctgaaaatgt aacaggactc 18060
tttaaagatt gtagtaaggt aatcactggg ttacatccta cacaggcacc tacacacctc 18120
agtgttgaca ctaaattcaa aactgaaggt ttatgtgttg acatacctgg catacctaag 18180
gacatgacct atagaagact catctctatg atgggtttta aaatgaatta tcaagttaat 18240
ggttacccta acatgtttat cacccgcgaa gaagctataa gacatgtacg tgcatggatt 18300
ggcttcgatg tcgaggggtg tcatgctact agagaagctg ttggtaccaa tttaccttta 18360
cagctaggtt tttctacagg tgttaaccta gttgctgtac ctacaggtta tgttgataca 18420
cctaataata cagatttttc cagagttagt gctaaaccac cgcctggaga tcaatttaaa 18480
cacctcatac cacttatgta caaaggactt ccttggaatg tagtgcgtat aaagattgta 18540
caaatgttaa gtgacacact taaaaatctc tctgacagag tcgtatttgt cttatgggca 18600
catggctttg agttgacatc tatgaagtat tttgtgaaaa taggacctga gcgcacctgt 18660
tgtctatgtg atagacgtgc cacatgcttt tccactgctt cagacactta tgcctgttgg 18720
catcattcta ttggatttga ttacgtctat aatccgttta tgattgatgt tcaacaatgg 18780
ggttttacag gtaacctaca aagcaaccat gatctgtatt gtcaagtcca tggtaatgca 18840
catgtagcta gttgtgatgc aatcatgact aggtgtctag ctgtccacga gtgctttgtt 18900
aagcgtgttg actggactat tgaatatcct ataattggtg atgaactgaa gattaatgcg 18960
gcttgtagaa aggttcaaca catggttgtt aaagctgcat tattagcaga caaattccca 19020
gttcttcacg acattggtaa ccctaaagct attaagtgtg tacctcaagc tgatgtagaa 19080
tggaagttct atgatgcaca gccttgtagt gacaaagctt ataaaataga agaattattc 19140
tattcttatg ccacacattc tgacaaattc acagatggtg tatgcctatt ttggaattgc 19200
aatgtcgata gatatcctgc taattccatt gtttgtagat ttgacactag agtgctatct 19260
aaccttaact tgcctggttg tgatggtggc agtttgtatg taaataaaca tgcattccac 19320
acaccagctt ttgataaaag tgcttttgtt aatttaaaac aattaccatt tttctattac 19380
tctgacagtc catgtgagtc tcatggaaaa caagtagtgt cagatataga ttatgtacca 19440
ctaaagtctg ctacgtgtat aacacgttgc aatttaggtg gtgctgtctg tagacatcat 19500
gctaatgagt acagattgta tctcgatgct tataacatga tgatctcagc tggctttagc 19560
ttgtgggttt acaaacaatt tgatacttat aacctctgga acacttttac aagacttcag 19620
agtttagaaa atgtggcttt taatgttgta aataagggac actttgatgg acaacagggt 19680
gaagtaccag tttctatcat taataacact gtttacacaa aagttgatgg tgttgatgta 19740
gaattgtttg aaaataaaac aacattacct gttaatgtag catttgagct ttgggctaag 19800
cgcaacatta aaccagtacc agaggtgaaa atactcaata atttgggtgt ggacattgct 19860
gctaatactg tgatctggga ctacaaaaga gatgctccag cacatatatc tactattggt 19920
gtttgttcta tgactgacat agccaagaaa ccaactgaaa cgatttgtgc accactcact 19980
gtcttttttg atggtagagt tgatggtcaa gtagacttat ttagaaatgc ccgtaatggt 20040
gttcttatta cagaaggtag tgttaaaggt ttacaaccat ctgtaggtcc caaacaagct 20100
agtcttaatg gagtcacatt aattggagaa gccgtaaaaa cacagttcaa ttattataag 20160
aaagttgatg gtgttgtcca acaattacct gaaacttact ttactcagag tagaaattta 20220
caagaattta aacccaggag tcaaatggaa attgatttct tagaattagc tatggatgaa 20280
ttcattgaac ggtataaatt agaaggctat gccttcgaac atatcgttta tggagatttt 20340
agtcatagtc agttaggtgg tttacatcta ctgattggac tagctaaacg ttttaaggaa 20400
tcaccttttg aattagaaga ttttattcct atggacagta cagttaaaaa ctatttcata 20460
acagatgcgc aaacaggttc atctaagtgt gtgtgttctg ttattgattt attacttgat 20520
gattttgttg aaataataaa atcccaagat ttatctgtag tttctaaggt tgtcaaagtg 20580
actattgact atacagaaat ttcatttatg ctttggtgta aagatggcca tgtagaaaca 20640
ttttacccaa aattacaatc tagtcaagcg tggcaaccgg gtgttgctat gcctaatctt 20700
tacaaaatgc aaagaatgct attagaaaag tgtgaccttc aaaattatgg tgatagtgca 20760
acattaccta aaggcataat gatgaatgtc gcaaaatata ctcaactgtg tcaatattta 20820
aacacattaa cattagctgt accctataat atgagagtta tacattttgg tgctggttct 20880
gataaaggag ttgcaccagg tacagctgtt ttaagacagt ggttgcctac gggtacgctg 20940
cttgtcgatt cagatcttaa tgactttgtc tctgatgcag attcaacttt gattggtgat 21000
tgtgcaactg tacatacagc taataaatgg gatctcatta ttagtgatat gtacgaccct 21060
aagactaaaa atgttacaaa agaaaatgac tctaaagagg gttttttcac ttacatttgt 21120
gggtttatac aacaaaagct agctcttgga ggttccgtgg ctataaagat aacagaacat 21180
tcttggaatg ctgatcttta taagctcatg ggacacttcg catggtggac agcctttgtt 21240
actaatgtga atgcgtcatc atctgaagca tttttaattg gatgtaatta tcttggcaaa 21300
ccacgcgaac aaatagatgg ttatgtcatg catgcaaatt acatattttg gaggaataca 21360
aatccaattc agttgtcttc ctattcttta tttgacatga gtaaatttcc ccttaaatta 21420
aggggtactg ctgttatgtc tttaaaagaa ggtcaaatca atgatatgat tttatctctt 21480
cttagtaaag gtagacttat aattagagaa aacaacagag ttgttatttc tagtgatgtt 21540
cttgttaaca actaaacgaa caatgtttgt ttttcttgtt ttattgccac tagtctctag 21600
tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac 21660
acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga 21720
cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggac 21780
caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc 21840
ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa 21900
gacccagtcc ctacttattg ttaataacgc tactaatgtt gttattaaag tctgtgaatt 21960
tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat 22020
ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca 22080
gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt 22140
gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt 22200
gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat 22260
taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga 22320
ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag 22380
gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact 22440
tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta 22500
tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac 22560
aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg 22620
gaacaggaag agaatcagca actgtgttgc tgattattct gtcctatata attccgcatc 22680
attttccact tttaagtgtt atggagtgtc tcctactaaa ttaaatgatc tctgctttac 22740
taatgtctat gcagattcat ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg 22800
gcaaactgga aagattgctg attataatta taaattacca gatgatttta caggctgcgt 22860
tatagcttgg aattctaaca atcttgattc taaggttggt ggtaattata attacctgta 22920
tagattgttt aggaagtcta atctcaaacc ttttgagaga gatatttcaa ctgaaatcta 22980
tcaggccggt agcacacctt gtaatggtgt tgaaggtttt aattgttact ttcctttaca 23040
atcatatggt ttccaaccca ctaatggtgt tggttaccaa ccatacagag tagtagtact 23100
ttcttttgaa cttctacatg caccagcaac tgtttgtgga cctaaaaagt ctactaattt 23160
ggttaaaaac aaatgtgtca atttcaactt caatggttta acaggcacag gtgttcttac 23220
tgagtctaac aaaaagtttc tgcctttcca acaatttggc agagacattg ctgacactac 23280
tgatgctgtc cgtgatccac agacacttga gattcttgac attacaccat gttcttttgg 23340
tggtgtcagt gttataacac caggaacaaa tacttctaac caggttgctg ttctttatca 23400
ggatgttaac tgcacagaag tccctgttgc tattcatgca gatcaactta ctcctacttg 23460
gcgtgtttat tctacaggtt ctaatgtttt tcaaacacgt gcaggctgtt taataggggc 23520
tgaacatgtc aacaactcat atgagtgtga catacccatt ggtgcaggta tatgcgctag 23580
ttatcagact cagactaatt ctcctcggcg ggcacgtagt gtagctagtc aatccatcat 23640
tgcctacact atgtcacttg gtgcagaaaa ttcagttgct tactctaata actctattgc 23700
catacccaca aattttacta ttagtgttac cacagaaatt ctaccagtgt ctatgaccaa 23760
gacatcagta gattgtacaa tgtacatttg tggtgattca actgaatgca gcaatctttt 23820
gttgcaatat ggcagttttt gtacacaatt aaaccgtgct ttaactggaa tagctgttga 23880
acaagacaaa aacacccaag aagtttttgc acaagtcaaa caaatttaca aaacaccacc 23940
aattaaagat tttggtggtt ttaatttttc acaaatatta ccagatccat caaaaccaag 24000
caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt 24060
catcaaacaa tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca 24120
aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata 24180
cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgc 24240
attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca 24300
gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa 24360
aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa 24420
ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat 24480
ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat 24540
tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc aacaattaat 24600
tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt 24660
acttggacaa tcaaaaagag ttgatttttg tggaaagggc tatcatctta tgtccttccc 24720
tcagtcagca cctcatggtg tagtcttctt gcatgtgact tatgtccctg cacaagaaaa 24780
gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg 24840
tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca 24900
aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt 24960
caacaacaca gtttatgatc ctttgcaacc tgaattagac tcattcaagg aggagttaga 25020
taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa 25080
tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt 25140
aaatgaatct ctcatcgatc tccaagaact tggaaagtat gagcagtata taaaatggcc 25200
atggtacatt tggctaggtt ttatagctgg cttgattgcc atagtaatgg tgacaattat 25260
gctttgctgt atgaccagtt gctgtagttg tctcaagggc tgttgttctt gtggatcctg 25320
ctgcaaattt gatgaagacg actctgagcc agtgctcaaa ggagtcaaat tacattacac 25380
ataaacgaac ttatggattt gtttatgaga atcttcacaa ttggaactgt aactttgaag 25440
caaggtgaaa tcaaggatgc tactccttca gattttgttc gcgctactgc aacgataccg 25500
atacaagcct cactcccttt cggatggctt attgttggcg ttgcacttct tgctgttttt 25560
cagagcgctt ccaaaatcat aaccctcaaa aagagatggc aactagcact ctccaagggt 25620
gttcactttg tttgcaactt gctgttgttg tttgtaacag tttactcaca ccttttgctc 25680
gttgctgctg gccttgaagc cccttttctc tatctttatg ctttagtcta cttcttgcag 25740
agtataaact ttgtaagaat aataatgagg ctttggcttt gctggaaatg ccgttccaaa 25800
aacccattac tttatgatgc caactatttt ctttgctggc atactaattg ttacgactat 25860
tgtatacctt acaatagtgt aacttcttca attgtcatta cttcaggtga tggcacaaca 25920
agtcctattt ctgaacatga ctaccagatt ggtggttata ctgaaaaatg ggaatctgga 25980
gtaaaagact gtgttgtatt acacagttac ttcacttcag actattacca gctgtactca 26040
actcaattga gtacagacac tggtgttgaa catgttacct tcttcatcta caataaaatt 26100
gttgatgagc ctgaagaaca tgtccaaatt cacacaatcg acggttcatc cggagttgtt 26160
aatccagtaa tggaaccaat ttatgatgaa ccgacgacga ctactagcgt gcctttgtaa 26220
gcacaagctg atgagtacga acttatgtac tcattcgttt cggaagagac aggtacgtta 26280
atagttaata gcgtacttct ttttcttgct ttcgtggtat tcttgctagt tacactagcc 26340
atccttactg cgcttcgatt gtgtgcgtac tgctgcaata ttgttaacgt gagtcttgta 26400
aaaccttctt tttacgttta ctctcgtgtt aaaaatctga attcttctag agttcctgat 26460
cttctggtct aaacgaacta aatattatat tagtttttct gtttggaact ttaattttag 26520
ccatggcaga ttccaacggt actattaccg ttgaagagct taaaaagctc cttgaacaat 26580
ggaacctagt aataggtttc ctattcctta catggatttg tcttctacaa tttgcctatg 26640
ccaacaggaa taggtttttg tatataatta agttaatttt cctctggctg ttatggccag 26700
taactttagc ttgttttgtg cttgctgctg tttacagaat aaattggatc accggtggaa 26760
ttgctatcgc aatggcttgt cttgtaggct tgatgtggct cagctacttc attgcttctt 26820
tcagactgtt tgcgcgtacg cgttccatgt ggtcattcaa tccagaaact aacattcttc 26880
tcaacgtgcc actccatggc actattctga ccagaccgct tctagaaagt gaactcgtaa 26940
tcggagctgt gatccttcgt ggacatcttc gtattgctgg acaccatcta ggacgctgtg 27000
acatcaagga cctgcctaaa gaaatcactg ttgctacatc acgaacgctt tcttattaca 27060
aattgggagc ttcgcagcgt gtagcaggtg actcaggttt tgctgcatac agtcgctaca 27120
ggattggcaa ctataaatta aacacagacc attccagtag cagtgacaat attgctttgc 27180
ttgtacagta agtgacaaca gatgtttcat ctcgttgact ttcaggttac tatagcagag 27240
atattactaa ttattatgag gacttttaaa gtttccattt ggaatcttga ttacatcata 27300
aacctcataa ttaaaaattt atctaagtca ctaactgaga ataaatattc tcaattagat 27360
gaagagcaac caatggagat tgattaaacg aacatgaaaa ttattctttt cttggcactg 27420
ataacactcg ctacttgtga gctttatcac taccaagagt gtgttagagg tacaacagta 27480
cttttaaaag aaccttgctc ttctggaaca tacgagggca attcaccatt tcatcctcta 27540
gctgataaca aatttgcact gacttgcttt agcactcaat ttgcttttgc ttgtcctgac 27600
ggcgtaaaac acgtctatca gttacgtgcc agatcagttt cacctaaact gttcatcaga 27660
caagaggaag ttcaagaact ttactctcca atttttctta ttgttgcggc aatagtgttt 27720
ataacacttt gcttcacact caaaagaaag acagaatgat tgaactttca ttaattgact 27780
tctatttgtg ctttttagcc tttctgctat tccttgtttt aattatgctt attatctttt 27840
ggttctcact tgaactgcaa gatcataatg aaacttgtca cgcctaaacg aacatgaaat 27900
ttcttgtttt cttaggaatc atcacaactg tagctgcatt tcaccaagaa tgtagtttac 27960
agtcatgtac tcaacatcaa ccatatgtag ttgatgaccc gtgtcctatt cacttctatt 28020
ctaaatggta tattagagta ggagctagaa aatcagcacc tttaattgaa ttgtgcgtgg 28080
atgaggctgg ttctaaatca cccattcagt acatcgatat cggtaattat acagtttcct 28140
gtttaccttt tacaattaat tgccaggaac ctaaattggg tagtcttgta gtgcgttgtt 28200
cgttctatga agacttttta gagtatcatg acgttcgtgt tgttttagat ttcatctaaa 28260
cgaacaaact aaaatgtctg ataatggacc ccaaaatcag cgaaatgcac cccgcattac 28320
gtttggtgga ccctcagatt caactggcag taaccagaat ggagaacgca gtggggcgcg 28380
atcaaaacaa cgtcggcccc aaggtttacc caataatact gcgtcttggt tcaccgctct 28440
cactcaacat ggcaaggaag accttaaatt ccctcgagga caaggcgttc caattaacac 28500
caatagcagt ccagatgacc aaattggcta ctaccgaaga gctaccagac gaattcgtgg 28560
tggtgacggt aaaatgaaag atctcagtcc aagatggtat ttctactacc taggaactgg 28620
gccagaagct ggacttccct atggtgctaa caaagacggc atcatatggg ttgcaactga 28680
gggagccttg aatacaccaa aagatcacat tggcacccgc aatcctgcta acaatgctgc 28740
aatcgtgcta caacttcctc aaggaacaac attgccaaaa ggcttctacg cagaagggag 28800
cagaggcggc agtcaagcct cttctcgttc ctcatcacgt agtcgcaaca gttcaagaaa 28860
ttcaactcca ggcagcagta ggggaacttc tcctgctaga atggctggca atggcggtga 28920
tgctgctctt gctttgctgc tgcttgacag attgaaccag cttgagagca aaatgtctgg 28980
taaaggccaa caacaacaag gccaaactgt cactaagaaa tctgctgctg aggcttctaa 29040
gaagcctcgg caaaaacgta ctgccactaa agcatacaat gtaacacaag ctttcggcag 29100
acgtggtcca gaacaaaccc aaggaaattt tggggaccag gaactaatca gacaaggaac 29160
tgattacaaa cattggccgc aaattgcaca atttgccccc agcgcttcag cgttcttcgg 29220
aatgtcgcgc attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc 29280
catcaaattg gatgacaaag atccaaattt caaagatcaa gtcattttgc tgaataagca 29340
tattgacgca tacaaaacat tcccaccaac agagcctaaa aaggacaaaa agaagaaggc 29400
tgatgaaact caagccttac cgcagagaca gaagaaacag caaactgtga ctcttcttcc 29460
tgctgcagat ttggatgatt tctccaaaca attgcaacaa tccatgagca gtgctgactc 29520
aactcaggcc taaactcatg cagaccacac aaggcagatg ggctatataa acgttttcgc 29580
ttttccgttt acgatatata gtctactctt gtgcagaatg aattctcgta actacatagc 29640
acaagtagat gtagttaact ttaatctcac atagcaatct ttaatcagtg tgtaacatta 29700
gggaggactt gaaagagcca ccacattttc accgaggcca cgcggagtac gatcgagtgt 29760
acagtgaaca atgctaggga gagctgccta tatggaagag ccctaatgtg taaaattaat 29820
tttagtagtg ctatccccat gtgattttaa tagcttctta ggagaatgac aaaaaaaaaa 29880
aaaaaaaaaa a 29891
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caatggttta acaggcacag g 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctcaagtgtc tgtggatcac g 21

Claims (1)

1. A SARS-CoV-2 adapted strain based on KI-hACE2 mouse, characterized in that: the full-length genome sequence is shown in SEQ ID NO. 1.
CN202210083596.0A 2022-01-25 2022-01-25 SARS-CoV-2 adaptive strain based on KI-hACE2 mouse and its construction method Active CN114107225B (en)

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WO2021213498A1 (en) * 2020-04-24 2021-10-28 中国食品药品检定研究院 Mouse model having knocked-in human ace2 gene, production method therefor and use thereof
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WO2021213498A1 (en) * 2020-04-24 2021-10-28 中国食品药品检定研究院 Mouse model having knocked-in human ace2 gene, production method therefor and use thereof
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CN112226464A (en) * 2020-09-16 2021-01-15 哈尔滨工业大学 Construction method and application of novel coronavirus humanized receptor hACE2 mouse model
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