质粒名称: | pCMV-PE2 |
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出品公司: | 爱迪基因 |
目录编号: | 132775 |
存储实验室: | David Liu |
载体骨架基本信息 |
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载体名称: | pCMV |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | -- |
完整质粒大小: | -- |
载体修饰: | 人细胞色素c氧化酶亚基VIII(human cytochrome C oxidase subunit VIII) |
载体类型: | 哺乳细胞表达 |
筛选标记: | 新霉素(G418) |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | PE2 |
物种来源: | -- |
插入大小: | 6354bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII(N端) |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | AgeI(未破坏) |
3' 克隆位点: | NotI (未破坏) |
5' 测序引物 | CMV-F |
3' 测序引物 | SV40pA-R |
文献引用方法 |
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材料和方法部分: | pCMV-PE2 was a gift from David Liu (Addgene plasmid # 132775 ; http://n2t.net/addgene:132775 ; RRID:Addgene_132775) |
参考文献部分: | Search-and-replace genome editing without double-strand breaks or donor DNA. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Nature. 2019 Oct 21. pii: 10.1038/s41586-019-1711-4. doi: 10.1038/s41586-019-1711-4. 10.1038/s41586-019-1711-4 PubMed 31634902 |
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载体基本信息
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载体序列
LOCUS pCMV-PE2 9753 bp ds-DNA circular SYN 18-OCT-2019 DEFINITION Prime editing in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-PE2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9753) TITLE Liu Lab plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 9753) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 18, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9753 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 694..714 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 715..4815 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(H840A) /note="generates RNA-guided single strand nicks in DNA due to the H840A mutation in the HNH catalytic domain" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6988..7008 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7017..7034 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(7057..7074) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7063..7287 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(7358..7374) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7358..7374) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7371..7393) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7382..7398 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(7406..7436) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7451..7472 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7589..7606) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7760..8348) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7840..7859) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8519..9379) /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 9142..9161 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9380..9484) /gene="bla" /label=AmpR promoter primer_bind complement(9563..9582) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc 601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg 661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcgacaag 721 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 781 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 841 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 901 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 961 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 1021 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 1081 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 1141 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 1201 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 1261 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 1321 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 1381 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 1441 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 1501 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 1561 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 1621 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 1681 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 1741 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 1801 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 1861 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 1921 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 1981 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 2041 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 2101 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 2161 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 2221 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 2281 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 2341 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 2401 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 2461 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 2521 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 2581 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 2641 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 2701 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 2761 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 2821 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 2881 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 2941 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 3001 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 3061 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 3121 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 3181 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc tatcgtgcct 3241 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 3301 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 3361 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 3421 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 3481 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 3541 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 3601 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 3661 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 3721 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 3781 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 3841 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 3901 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 3961 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 4021 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 4081 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 4141 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 4201 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 4261 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 4321 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 4381 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 4441 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 4501 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 4561 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 4621 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 4681 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 4741 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 4801 cagctgggag gtgactctgg aggatctagc ggaggatcct ctggcagcga gacaccagga 4861 acaagcgagt cagcaacacc agagagcagt ggcggcagca gcggcggcag cagcacccta 4921 aatatagaag atgagtatcg gctacatgag acctcaaaag agccagatgt ttctctaggg 4981 tccacatggc tgtctgattt tcctcaggcc tgggcggaaa ccgggggcat gggactggca 5041 gttcgccaag ctcctctgat catacctctg aaagcaacct ctacccccgt gtccataaaa 5101 caatacccca tgtcacaaga agccagactg gggatcaagc cccacataca gagactgttg 5161 gaccagggaa tactggtacc ctgccagtcc ccctggaaca cgcccctgct acccgttaag 5221 aaaccaggga ctaatgatta taggcctgtc caggatctga gagaagtcaa caagcgggtg 5281 gaagacatcc accccaccgt gcccaaccct tacaacctct tgagcgggct cccaccgtcc 5341 caccagtggt acactgtgct tgatttaaag gatgcctttt tctgcctgag actccacccc 5401 accagtcagc ctctcttcgc ctttgagtgg agagatccag agatgggaat ctcaggacaa 5461 ttgacctgga ccagactccc acagggtttc aaaaacagtc ccaccctgtt taatgaggca 5521 ctgcacagag acctagcaga cttccggatc cagcacccag acttgatcct gctacagtac 5581 gtggatgact tactgctggc cgccacttct gagctagact gccaacaagg tactcgggcc 5641 ctgttacaaa ccctagggaa cctcgggtat cgggcctcgg ccaagaaagc ccaaatttgc 5701 cagaaacagg tcaagtatct ggggtatctt ctaaaagagg gtcagagatg gctgactgag 5761 gccagaaaag agactgtgat ggggcagcct actccgaaga cccctcgaca actaagggag 5821 ttcctaggga aggcaggctt ctgtcgcctc ttcatccctg ggtttgcaga aatggcagcc 5881 cccctgtacc ctctcaccaa accggggact ctgtttaatt ggggcccaga ccaacaaaag 5941 gcctatcaag aaatcaagca agctcttcta actgccccag ccctggggtt gccagatttg 6001 actaagccct ttgaactctt tgtcgacgag aagcagggct acgccaaagg tgtcctaacg 6061 caaaaactgg gaccttggcg tcggccggtg gcctacctgt ccaaaaagct agacccagta 6121 gcagctgggt ggcccccttg cctacggatg gtagcagcca ttgccgtact gacaaaggat 6181 gcaggcaagc taaccatggg acagccacta gtcattctgg ccccccatgc agtagaggca 6241 ctagtcaaac aaccccccga ccgctggctt tccaacgccc ggatgactca ctatcaggcc 6301 ttgcttttgg acacggaccg ggtccagttc ggaccggtgg tagccctgaa cccggctacg 6361 ctgctcccac tgcctgagga agggctgcaa cacaactgcc ttgatatcct ggccgaagcc 6421 cacggaaccc gacccgacct aacggaccag ccgctcccag acgccgacca cacctggtac 6481 acggatggaa gcagtctctt acaagaggga cagcgtaagg cgggagctgc ggtgaccacc 6541 gagaccgagg taatctgggc taaagccctg ccagccggga catccgctca gcgggctgaa 6601 ctgatagcac tcacccaggc cctaaagatg gcagaaggta agaagctaaa tgtttatact 6661 gatagccgtt atgcttttgc tactgcccat atccatggag aaatatacag aaggcgtggg 6721 tggctcacat cagaaggcaa agagatcaaa aataaagacg agatcttggc cctactaaaa 6781 gccctctttc tgcccaaaag acttagcata atccattgtc caggacatca aaagggacac 6841 agcgccgagg ctagaggcaa ccggatggct gaccaagcgg cccgaaaggc agccatcaca 6901 gagactccag acacctctac cctcctcata gaaaattcat caccctctgg cggctcaaaa 6961 agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcta accggtcatc 7021 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc 7081 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 7141 ctgtcctttc ctaataaaat gagaaaattg catcgcattg tctgagtagg tgtcattcta 7201 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 7261 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga 7321 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 7381 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 7441 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 7501 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 7561 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 7621 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 7681 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 7741 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 7801 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 7861 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 7921 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 7981 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 8041 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 8101 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 8161 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 8221 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 8281 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 8341 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact 8401 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 8461 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 8521 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 8581 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 8641 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 8701 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 8761 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 8821 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 8881 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 8941 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 9001 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 9061 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 9121 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 9181 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 9241 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 9301 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 9361 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 9421 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 9481 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg 9541 atcccctagg gtctactctc agtacaatct gctctgatgc cgcatagtta agccagtatc 9601 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa 9661 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc 9721 tgcttcgcga tgtacgggcc agatatacgc gtt //