质粒名称: | pEMPTY::sgRNA2 |
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目录编号: | 165459 |
存储实验室: | Inigo Lasa |
载体骨架基本信息 |
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载体名称: | pCN38, pMAD |
完整质粒大小: | 11277 bp |
载体类型: | Bacterial Expression, CRISPR, Synthetic Biology |
筛选标记: | In Gram-positive bacteria: chloramphenicol |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | gRNA / shRNA |
物种来源: | Staphylococcus aureus |
启动子: | SP01 |
克隆方法: | 限制性内切酶 |
5’克隆位点: | AscI(未破坏) |
3’克隆位点: | SmaI (未破坏) |
5’测序引物 | cgatttttgtgatgctcgtcaggg |
3’测序引物 | cgtaaacggatgctggctag |
文献引用方法 |
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材料和方法部分: | pEMPTY::sgRNA2 was a gift from Inigo Lasa (Addgene plasmid # 165459 ; http://n2t.net/addgene:165459 ; RRID:Addgene_165459) |
参考文献部分: | Fitness Cost Evolution of Natural Plasmids of Staphylococcus aureus. Dorado-Morales P, Garcillan-Barcia MP, Lasa I, Solano C. mBio. 2021 Feb 23;12(1). pii: mBio.03094-20. doi: 10.1128/mBio.03094-20. 10.1128/mBio.03094-20 PubMed 33622733 |
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载体序列
LOCUS pEMPTY::sgRNA2 11278 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Escherichia coli – Staphylococcus aureus shuttle vector for plasmid curing in Gram-positive bacteria. ACCESSION . VERSION . KEYWORDS pEMPTY::sgRNA2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11278) AUTHORS Dorado-Morales P, Garcillan-Barcia MP, Lasa I, Solano C TITLE Fitness Cost Evolution of Natural Plasmids of Staphylococcus aureus. JOURNAL mBio. 2021 Feb 23;12(1). pii: mBio.03094-20. doi: 10.1128/mBio.03094-20. PUBMED 33622733 REFERENCE 2 (bases 1 to 11278) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..11278 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1176..1198 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(1236..1254) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1322..1426 /gene="bla" /label=AmpR promoter CDS 1427..2287 /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(1645..1664) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2458..3046 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2947..2966 /label=pBR322ori-F /note="pBR322 origin, forward primer" misc_RNA 3260..3335 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 3416..3463 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 3459..3477 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(3512..7618) /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" protein_bind 7661..7679 /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O1 for the tetR and tetA genes" CDS 7789..8412 /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind complement(7862..7881) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" primer_bind complement(8493..8510) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(8493..8509) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8502..8524) /label=M13/pUC Forward /note="In lacZ gene" ORIGIN 1 ataggcttgg ttatgccggt actgccgggc ctcttgcggg atcgaggggg cccacctagg 61 tattatcaag ataagaaaga aaaggatttt tcgctacgct caaatccttt aaaaaaacac 121 aaaagaccac attttttaat gtggtctttt attcttcaac taaagcaccc attagttcaa 181 caaacgaaaa ttggataaag tgggatattt ttaaaatata tatttatgtt acagtaatat 241 tgacttttaa aaaaggattg attctaatga agaaagcaga caagtaagcc tcctaaattc 301 actttagata aaaatttagg aggcatatca aatgaacttt aataaaattg atttagacaa 361 ttggaagaga aaagagatat ttaatcatta tttgaaccaa caaacgactt ttagtataac 421 cacagaaatt gatattagtg ttttataccg aaacataaaa caagaaggat ataaatttta 481 ccctgcattt attttcttag tgacaagggt gataaactca aatacagctt ttagaactgg 541 ttacaatagc gacggagagt taggttattg ggataagtta gagccacttt atacaatttt 601 tgatggtgta tctaaaacat tctctggtat ttggactcct gtaaagaatg acttcaaaga 661 gttttatgat ttataccttt ctgatgtaga gaaatataat ggttcgggga aattgtttcc 721 caaaacacct atacctgaaa atgctttttc tctttctatt attccatgga cttcatttac 781 tgggtttaac ttaaatatca ataataatag taattacctt ctacccatta ttacagcagg 841 aaaattcatt aataaaggta attcaatata tttaccgcta tctttacagg tacatcattc 901 tgtttgtgat ggttatcatg caggattgtt tatgaactct attcaggaat tgtcagatag 961 gcctaatgac tggcttttat aatatgagat aatgccgact gtacttttta cagtcggttt 1021 tctaatgtca ctaacctgcc ccgttagttg aagaaggccg cggcctcgag cggccgcata 1081 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 1141 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 1201 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 1261 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 1321 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 1381 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 1441 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 1501 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 1561 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 1621 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 1681 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 1741 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 1801 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 1861 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 1921 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 1981 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 2041 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 2101 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 2161 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 2221 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 2281 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 2341 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 2401 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 2461 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2521 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2581 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 2641 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2701 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2761 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2821 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 2881 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 2941 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 3001 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 3061 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 3121 atcccctgat tctgtggata accgtattac cgcctttgag tgagctggcg gccgctgcat 3181 gcctgcaggg cgcgccttga caaattgcag taggcatgac aaaatggact cacaagtttt 3241 gggattgtta agggttccgg ttttagagct agaaatagca agttaaaata aggctagtcc 3301 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcgcaaaaa accccgcttc 3361 ggcggggttt tttcgctagt cggtgtgcgc aaccttaagc ccgggtacca gatctcaaaa 3421 aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc tcagcggtgg 3481 cagcagccta ggataccggt aacatgggat ctcagtcacc tcctagctga ctcaaatcaa 3541 tgcgtgtttc ataaagacca gtgatggatt gatggataag agtggcatct aaaacttctt 3601 ttgtagacgt atatcgttta cgatcaattg ttgtatcaaa atatttaaaa gcagcgggag 3661 ctccaagatt cgtcaacgta aataaatgaa taatattttc tgcttgttca cgtattggtt 3721 tgtctctatg tttgttatat gcactaagaa ctttatctaa attggcatct gctaaaataa 3781 cacgcttaga aaattcactg atttgctcaa taatctcatc taaataatgc ttatgctgct 3841 ccacaaacaa ttgtttttgt tcgttatctt ctggactacc cttcaacttt tcataatgac 3901 tagctaaata taaaaaattc acatatttgc ttggcagagc cagctcattt cctttttgta 3961 attctccggc actagccagc atccgtttac gaccgttttc taactcaaaa agactatatt 4021 taggtagttt aatgattaag tcttttttaa cttccttata tcctttagct tctaaaaagt 4081 caatcggatt tttttcaaag gaacttcttt ccataattgt gatccctagt aactctttaa 4141 cggattttaa cttcttcgat ttcccttttt ccaccttagc aaccactagg actgaataag 4201 ctaccgttgg actatcaaaa ccaccatatt tttttggatc ccagtctttt ttacgagcaa 4261 taagcttgtc cgaatttctt tttggtaaaa ttgactcctt ggagaatccg cctgtctgta 4321 cttctgtttt cttgacaata ttgacttggg gcatggacaa tactttgcgc actgtggcaa 4381 aatctcgccc tttatcccag acaatttctc cagtttcccc attagtttcg attagagggc 4441 gtttgcgaat ctctccattt gcaagtgtaa tttctgtttt gaagaagttc atgatattag 4501 agtaaaagaa atattttgcg gttgctttgc ctatttcttg ctcagactta gcaatcattt 4561 tacgaacatc ataaacttta taatcaccat agacaaactc cgattcaagt tttggatatt 4621 tcttaatcaa agcagttcca acgacggcat ttagatacgc atcatgggca tgatggtaat 4681 tgttaatctc acgtacttta tagaattgga aatcttttcg gaagtcagaa actaatttag 4741 attttaaggt aatcacttta acctctcgaa taagtttatc attttcatcg tatttagtat 4801 tcatgcgact atccaaaatt tgtgccacat gcttagtgat ttggcgagtt tcaaccaatt 4861 ggcgtttgat aaaaccagct ttatcaagtt cactcaaacc tccacgttca gctttcgtta 4921 aattatcaaa cttacgttga gtgattaact tggcgtttag aagttgtctc caatagtttt 4981 tcatcttttt gactacttct tcacttggaa cgttatccga tttaccacga tttttatcag 5041 aacgcgttaa gaccttattg tctattgaat cgtctttaag gaaactttgt ggaacaatgt 5101 gatcgacatc ataatcactt aaacgattaa tatctaattc ttggtccaca tacatgtctc 5161 ttccattttg gagataatag agatagagct tttcattttg caattgagta ttttcaacag 5221 gatgctcttt aagaatctga cttcctaatt ctttgatacc ttcttcgatt cgtttcatac 5281 gctctcgcga atttttctgg cccttttgag ttgtctgatt ttcacgtgcc atttcaataa 5341 cgatattttc tggcttatgc cgccccatta ctttgaccaa ttcatcaaca acttttacag 5401 tctgtaaaat acctttttta atagcagggc taccagctaa atttgcaata tgttcatgta 5461 aactatcgcc ttgtccagac acttgtgctt tttgaatgtc ttctttaaat gtcaaactat 5521 catcatggat cagctgcata aaattgcgat tggcaaaacc atctgatttc aaaaaatcta 5581 atattgtttt gccagattgc ttatccctaa taccattaat caattttcga gacaaacgtc 5641 cccaaccagt ataacggcga cgtttaagct gtttcatcac cttatcatca aagaggtgag 5701 catatgtttt aagtctttcc tcaatcatct ccctatcttc aaataaggtc aatgttaaaa 5761 caatatcctc taagatatct tcattttctt cattatccaa aaaatcttta tctttaataa 5821 tttttagcaa atcatggtag gtacctaatg aagcattaaa tctatcttca actcctgaaa 5881 tttcaacact atcaaaacat tctatttttt tgaaataatc ttcttttaat tgcttaacgg 5941 ttacttttcg atttgttttg aagagtaaat caacaatggc tttcttctgt tcacctgaaa 6001 gaaatgctgg ttttcgcatt ccttcagtaa catatttgac ctttgtcaat tcgttataaa 6061 ccgtaaaata ctcataaagc aaactatgtt ttggtagtac tttttcattt ggaagatttt 6121 tatcaaagtt tgtcatgcgt tcaataaatg attgagctga agcaccttta tcgacaactt 6181 cttcaaaatt ccatggggta attgtttctt cagacttccg agtcatccat gcaaaacgac 6241 tattgccacg cgccaatgga ccaacataat aaggaattcg aaaagtcaag attttttcaa 6301 tcttctcacg attgtctttt aaaaatggat aaaagtcttc ttgtcttctc aaaatagcat 6361 gcagctcacc caagtgaatt tgatggggaa tagagccgtt gtcaaaggtc cgttgcttgc 6421 gcagcaaatc ttcacgattt agtttcacca ataattcctc agtaccatcc attttttcta 6481 aaattggttt gataaattta taaaattctt cttggctagc tcccccatca atataacctg 6541 catatccgtt ttttgattga tcaaaaaaga tttctttata cttttctgga agttgttgtc 6601 gaactaaagc ttttaaaaga gtcaagtctt gatgatgttc atcgtagcgt ttaatcattg 6661 aagctgatag gggagcctta gttatttcag tatttactct taggatatct gaaagtaaaa 6721 tagcatctga taaattctta gctgccaaaa acaaatcagc atattgatct ccaatttgcg 6781 ccaataaatt atctaaatca tcatcgtaag tatcttttga aagctgtaat ttagcatctt 6841 ctgccaaatc aaaatttgat ttaaaattag gggtcaaacc caatgacaaa gcaatgagat 6901 tcccaaataa gccatttttc ttctcaccgg ggagctgagc aatgagattt tctaatcgtc 6961 ttgatttact caatcgtgca gaaagaatcg ctttagcatc tactccactt gcgttaatag 7021 ggttttcttc aaataattga ttgtaggttt gtaccaactg gataaatagt ttgtccacat 7081 cactattatc aggatttaaa tctccctcaa tcaaaaaatg accacgaaac ttaatcatat 7141 gcgctaaggc caaatagatt aagcgcaaat ccgctttatc agtagaatct accaattttt 7201 ttcgcagatg atagatagtt ggatatttct catgataagc aacttcatct actatatttc 7261 caaaaatagg atgacgttca tgcttcttgt cttcttccac caaaaaagac tcttcaagtc 7321 gatgaaagaa actatcatct actttcgcca tctcatttga aaaaatctcc tgtagataac 7381 aaatacgatt cttccgacgt gtataccttc tacgagctgt ccgtttgaga cgagtcgctt 7441 ccgctgtctc tccactgtca aataaaagag cccctataag attttttttg atactgtggc 7501 ggtctgtatt tcccagaacc ttgaactttt tagacggaac cttatattca tcagtgatca 7561 ccgcccatcc gacgctattt gtgccgatat ctaagcctat tgagtatttc ttatccataa 7621 ataatcatcc tcctaaagat ctcaagctta ttttaattat actctatcaa tgatagagtg 7681 tcaatatttt ttttagtttt tcatgaactc gaggggatcc aaataaaaaa ctagtttgac 7741 aaataactct atcaatgata taatgtcaac aaaaaggagg aattaatgat gtctagatta 7801 gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt 7861 ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat 7921 gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat 7981 actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa 8041 agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg 8101 cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt 8161 ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc 8221 gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat 8281 agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca 8341 gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa 8401 agtgggtctt aaaagcagca taaccttttt ccgtgatggt aacttcacgg taaccaagat 8461 gtcgagttat cgataccgtc gacctggaat tcactggccg tcgttttaca acgtcgtgac 8521 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 8581 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 8641 ggcgaatggc gcctgagggt tgccagagtt aaaggatcaa gtaatacaaa cgggatacaa 8701 agacataatc aaagagagaa taaaaactat aataataaag acataaatca tgaggaaaca 8761 tataaaaatt atgatttgat taacgcacaa aatataaagt ataaagataa aattgatgaa 8821 acgattgatg agaattattc agggaaacgt aaaattcggt cagatgcaat tcgacatgtg 8881 gacggactgg ttacaagtga taaagatttc tttgatgatt taagcggaga agaaatagaa 8941 cgatttttta aagatagctt ggagtttcta gaaaatgaat acggtaagga aaatatgctg 9001 tatgcgactg tccatctgga tgaaagagtc ccacatatgc actttggttt tgtcccttta 9061 acagaggacg ggagattgtc tgcaaaagaa cagttaggca acaagaaaga ctttactcaa 9121 ttacaagata gatttaatga gtatgtgaat gagaaaggtt atgaacttga aagaggcacg 9181 tccaaagagg ttacagaacg agaacataaa gcgatggatc agtacaagaa agatactgta 9241 tttcataaac aggaactgca agaagttaag gatgagttac agaaggcaaa taagcagtta 9301 cagagtggaa tagagcatat gaggtctacg aaaccctttg attatgaaaa tgagcgtaca 9361 ggtttgttct ctggacgtga agagactggt agaaagatat taactgctga tgaatttgaa 9421 cgcctgcaag aaacaatctc ttctgcagaa cggattgttg atgattacga aaatattaag 9481 agcacagact attacacaga aaatcaagaa ttaaaaaaac gtagagagag tttgaaagaa 9541 gtagtgaata catggaaaga ggggtatcac gaaaaaagta aagaggttaa taaattaaag 9601 cgagagaatg atagtttgaa tgagcagttg aatgtatcag agaaatttca agatagtaca 9661 gtgactttat atcgtgctgc gagggcgaat ttccctgggt ttgagaaagg gtttaatagg 9721 cttaaagaga aattctttaa tgattccaaa ttcgagcgtg tgggacagtt tatggatgtt 9781 gtacaggata atgtccagaa ggtcgataga aagcgtgaga aacagcgtac agacgattta 9841 gagatgtaga ggtactttta tgccgagaaa actttttgcg tgtgacagtc cttaaaatat 9901 acttagagcg taagcgaaag tagtagcgac agctattaac tttcggttgc aaagctctag 9961 gatttttaat ggacgcagcg catcacacgc aaaaaggaaa ttggaataaa tgcgaaattt 10021 gagatgttaa ttaaagacct ttttgaggtc tttttttctt agatttttgg ggttatttag 10081 gggagaaaac ataggggggt actacgacct cccccctagg tgtccattgt ccattgtcca 10141 aacaaataaa taaatattgg gtttttaatg ttaaaaggtt gttttttatg ttaaagtgaa 10201 aaaaacagat gttgggaggt acagtgatgg ttgtagatag aaaagaagag aaaaaagttg 10261 ctgttacttt aagacttaca acagaagaaa atgagatatt aaatagaatc aaagaaaaat 10321 ataatattag caaatcagat gcaaccggta ttctaataaa aaaatatgca aaggaggaat 10381 acggtgcatt ttaaacaaaa aaagatagac agcactggca tgctgcctat ctatgactaa 10441 attttgttaa atgtattagc accgttatta tatcatgagc gaaaatgtaa taaaagaaac 10501 tgaaaacaag aaaaattcaa gaggacgtaa ttggacattt gttttatatc cagaatcagc 10561 aaaagccgag tggttagagt atttaaaaga gttacacatt caatttgtag tgtctccatt 10621 acatgatagg gatactgata cagaagatag gatgaaaaaa gagcattatc atattctagt 10681 gatgtatgag ggtaataaat cttatgaaca gataaaaata attacagaag aattgaatgc 10741 gactattccg cagattgcag gaagtgtgaa aggtcttgtg agatatatgc ttcacatgga 10801 cgatcctaat aaatttaaat atcaaaaaga agatatgata gtttatggcg gtgtagatgt 10861 tgatgaatta ttaaagaaaa caacaacaga tagatataaa ttaattaaag aaatgattga 10921 gtttattgat gaacaaggaa tcgtagaatt taagagttta atggattatg caatgaagtt 10981 taaatttgat gattggttcc cgcttttatg tgataactcg gcgtatgtta ttcaagaata 11041 tataaaatca aatcggtata aatctgaccg atagattttg aatttaagag tgtcacaaga 11101 cactcttttt tcgcaccaac gaaaactggt ttaagccgac tgcgcaaaag acataatcga 11161 taagctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg caccgtgtat 11221 gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg ctgtaggc //