质粒名称: | pspCas9-POLI-ST2-com vector |
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出品公司: | Addgene |
目录编号: | 176234 |
存储实验室: | Baisong Lu |
载体骨架基本信息 |
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载体名称: | Addgene# 136269 |
完整质粒大小: | 11588bp |
载体类型: | 细菌细胞表达 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 未知 |
文献引用方法 |
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材料和方法部分: |
pspCas9-POLI-ST2-com vector was a gift from Baisong Lu (Addgene plasmid # 176234 ; http://n2t.net/addgene:176234 ; RRID:Addgene_176234)
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参考文献部分: | Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9. Yoo KW, Yadav MK, Song Q, Atala A, Lu B. Nucleic Acids Res. 2022 Mar 22. pii: 6552087. doi: 10.1093/nar/gkac186. 10.1093/nar/gkac186 PubMed 35323942 |
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载体基本信息
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载体序列
LOCUS pspCas9-POLI-ST2-com vector 11588 bp ds-DNA circular SYN 29-NOV-2021 DEFINITION Vector plasmid for expressing spCas9-POLI fusion protein and sgRNA. There are two copies of HBB 3' UTR in the 3'UTR of spCas9-POLI to enhance expression. The ST2 loop of the sgRNA scaffold was replace. ACCESSION . VERSION . KEYWORDS pspCas9-POLI-ST2-com vector SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11588) TITLE Targeting DNA Polymerase to DNA double strand breaks reduced the sizes of CRISPR/Cas9-induced DNA deletions in human cells JOURNAL Unpublished REFERENCE 2 (bases 1 to 11588) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 29, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..11588 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 6..291 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" intron 571..798 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 810..819 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 819..884 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 891..911 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 936..5036 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5037..5084 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" RBS 5867..5875 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" primer_bind complement(8247..8264) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 8253..8460 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 8472..8612 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 8472..8601 /label=AAV2 ITR rep_origin 8687..9142 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8774..8793) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8984..9005 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(9159..9178) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 9278..9300 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(9338..9356) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 9424..9528 /gene="bla" /label=AmpR promoter CDS 9529..10389 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(9747..9766) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 10560..11148 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 11049..11068 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 11210..11450 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 11210..11230 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 11381..11400 /label=LKO.1 5' /note="Human U6 promoter, forward primer" ORIGIN 1 gtacccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 61 ccattgacgt caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 121 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 181 attgacgtca atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg 241 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg 301 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat 361 ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg 421 ccrggsgggg sggggggggg sggggggsgg ggsggggsgr ggsggrgrgg kgcggcggca 481 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 541 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg 601 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca 661 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa 721 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct 781 gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac cacgacggag 841 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga 901 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg 961 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca 1021 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag 1081 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa 1141 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga 1201 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg 1261 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg 1321 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg 1381 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga 1441 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc 1501 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg 1561 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc 1621 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc 1681 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca 1741 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt 1801 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca 1861 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc 1921 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga 1981 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg 2041 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc 2101 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca 2161 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt 2221 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct 2281 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg 2341 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga 2401 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca 2461 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg 2521 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg 2581 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca 2641 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct 2701 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg 2761 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag 2821 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc 2881 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca 2941 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca 3001 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga 3061 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca 3121 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag 3181 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca 3241 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag 3301 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga 3361 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca 3421 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg 3481 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg 3541 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca 3601 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg 3661 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc 3721 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga 3781 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt 3841 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga 3901 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt 3961 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg 4021 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga 4081 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg 4141 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc 4201 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta 4261 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga 4321 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg 4381 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg 4441 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag 4501 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc 4561 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct 4621 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg 4681 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca 4741 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag 4801 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca 4861 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca 4921 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc 4981 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa 5041 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc tctgagacac 5101 caggcacctc cgagtctgcc acccctgagg gaggaagcgg aggaagcggc tccgtgcaga 5161 tcccacagaa ccccctgatc ctggtggacg gcagctccta cctgtatcgg gcctaccacg 5221 ccttcccacc tctgacaaac tccgccggag agccaaccgg agccatgtat ggcgtgctga 5281 atatgctgag gagcctgatc atgcagtaca agcctacaca cgccgccgtg gtgtttgatg 5341 ccaagggcaa gaccttccgc gacgagctgt ttgagcacta caagagccac aggccaccaa 5401 tgcctgacga tctgagggca cagatcgagc cactgcacgc aatggtgaag gccatgggcc 5461 tgcctctgct ggccgtgagc ggagtggagg ccgacgatgt gatcggcaca ctggcaaggg 5521 aggcagagaa ggcaggccgc ccagtgctga tctccaccgg cgacaaggat atggcccagc 5581 tggtgacacc aaacatcacc ctgatcaaca ccatgacaaa taccatcctg ggccccgagg 5641 aggtggtgaa taagtatggc gtgcctccag agctgatcat cgatttcctg gccctgatgg 5701 gcgactctag cgataacatc cctggcgtgc caggagtggg agaaaagacc gcacaggccc 5761 tgctgcaggg cctgggaggc ctggacaccc tgtacgccga gccagagaag atcgccggcc 5821 tgtcctttag gggcgccaag acaatggccg ccaagctgga gcagaataag gaggtggcct 5881 acctgtctta tcagctggcc acaatcaaga ccgacgtgga gctggagctg acctgcgagc 5941 agctggaggt gcagcagcct gcagcagagg agctgctggg cctgttcaag aagtacgagt 6001 ttaagagatg gacagccgat gtggaggccg gcaagtggct gcaggcaaag ggagcaaagc 6061 cagcagcaaa gccacaggag acaagcgtgg cagacgaggc accagaggtg acagccaccg 6121 tgatctccta cgataactat gtgacaatcc tggacgagga gacactgaag gcctggatcg 6181 ccaagctgga gaaggccccc gtgttcgcct ttgatacaga gacagacagc ctggataaca 6241 tctccgccaa tctggtgggc ctgtctttcg ccatcgagcc tggcgtggcc gcctatatcc 6301 cagtggccca cgactacctg gatgcccccg accagatcag cagggagaga gccctggagc 6361 tgctgaagcc tctgctggag gatgagaagg ccctgaaggt cggccagaac ctgaagtatg 6421 acaggggcat cctggccaat tacggcatcg agctgagagg catcgccttt gacaccatgc 6481 tggagtctta tatcctgaat agcgtggcag gccggcacga catggattcc ctggccgaga 6541 ggtggctgaa gcacaagaca atcaccttcg aggagatcgc cggcaagggc aagaaccagc 6601 tgaccttcaa ccagatcgcc ctggaggagg caggcaggta cgcagcagag gacgcagatg 6661 tgaccctgca gctgcacctg aagatgtggc cagatctgca gaagcacaag ggccccctga 6721 acgtctttga gaatatcgag atgcccctgg tgcctgtgct gagccggatc gagcgcaacg 6781 gcgtgaagat cgaccctaag gtgctgcaca atcactccga ggagctgacc ctgagactgg 6841 ccgagctgga gaagaaggcc cacgagatcg ccggcgagga gttcaacctg tcctctacaa 6901 agcagctgca gaccatcctg tttgagaagc agggcatcaa gcccctgaag aaaacccctg 6961 gaggagcacc atctaccagc gaggaggtgc tggaggagct ggccctggat tatcccctgc 7021 ctaaagtgat cctggagtac cggggcctgg ccaagctgaa gtctacatat accgacaagc 7081 tgcccctgat gatcaaccct aagacaggcc gggtgcacac cagctaccac caggcagtga 7141 cagcaaccgg ccgcctgagc tccaccgatc caaacctgca gaatatcccc gtgaggaatg 7201 aggagggcag gagaatcaga caggccttca tcgcccccga ggattatgtg atcgtgtccg 7261 ccgactactc tcagatcgag ctgaggatca tggcccacct gtccagagat aagggcctgc 7321 tgacagcctt cgccgagggc aaggacatcc acagggcaac cgcagcagag gtgtttggcc 7381 tgcctctgga gacagtgacc tccgagcagc ggcgctctgc caaggccatc aacttcggcc 7441 tgatctatgg catgtctgcc tttggcctgg ccaggcagct gaatatccct agaaaggagg 7501 cccagaagta catggacctg tatttcgaga gataccccgg cgtgctggag tacatggaga 7561 ggacacgcgc acaggcaaag gagcagggct atgtggagac actggatggc aggagactgt 7621 acctgccaga catcaagtct agcaacggag caaggagggc agcagcagag agggccgcca 7681 tcaatgcccc catgcagggc acagccgccg atatcatcaa gagagccatg atcgcagtgg 7741 acgcctggct gcaggcagag cagccaaggg tgagaatgat catgcaggtg cacgatgagc 7801 tggtgtttga ggtgcacaag gacgatgtgg acgccgtggc caagcagatc caccagctga 7861 tggagaactg tacccgcctg gatgtgccac tgctggtgga agtgggaagc ggagagaatt 7921 gggaccaggc ccactgagga tcctaagctc gctttcttgc tgtccaattt ctattaaagg 7981 ttcctttgtt ccctaagtcc aactactaaa ctgggggata ttatgaaggg ccttgagcat 8041 ctggattctg cctaataaaa aacatttatt ttcattgcta gctcgctttc ttgctgtcca 8101 atttctatta aaggttcctt tgttccctaa gtccaactac taaactgggg gatattatga 8161 agggccttga gcatctggat tctgcctaat aaaaaacatt tattttcatt gcgaattcta 8221 gaaaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 8281 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 8341 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 8401 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 8461 ggtacggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 8521 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 8581 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 8641 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 8701 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 8761 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 8821 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 8881 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 8941 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 9001 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg 9061 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 9121 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 9181 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 9241 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 9301 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 9361 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 9421 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 9481 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 9541 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 9601 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 9661 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 9721 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 9781 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 9841 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 9901 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 9961 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 10021 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 10081 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 10141 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 10201 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 10261 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 10321 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 10381 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 10441 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 10501 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 10561 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 10621 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 10681 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc 10741 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 10801 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 10861 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 10921 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 10981 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 11041 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 11101 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 11161 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt 11221 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat 11281 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt 11341 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt 11401 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg 11461 tgtcttcctc gaggaagacc cgtttgagag ctagaaatag caagttcaaa taaggctagt 11521 ccgttatcaa cttggctgaa tgcctgcgag catcccaccc aagtggcacc gagtcggtgc 11581 tttttttg //
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