LOCUS Exported 11106 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION All-in-one CRISPR plasmid for integrating, deleting, or replacing a gene in S. cerevisiae. High-copy, URA3 plasmid contains human codon-optimized Cas9 and a guide RNA targeting ARS106a sequence ATACGGTCAGGGTAGCGCCC in yeast chromosome 1.. ACCESSION . VERSION . KEYWORDS p426_Cas9_gRNA-ARS106a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11106) AUTHORS Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A TITLE A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. JOURNAL Nucleic Acids Res. 2017 Jan 9;45(1):496-508. doi: 10.1093/nar/gkw1023. Epub 2016 Nov 28. PUBMED 27899650 REFERENCE 2 (bases 1 to 11106) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11106 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 81..100 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 334..351 /label=L4440 /note="L4440 vector, forward primer" protein_bind 468..489 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 504..534 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 542..558 /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)." primer_bind 547..569 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 566..582 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 566..582 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 601..621 /label=T3 /note="T3 promoter, forward primer" promoter 603..621 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_RNA 848..923 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 1009..1713 /gene="S. cerevisiae ADH1" /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" CDS 1723..5826 /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 FLVEEDKKNERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIYLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5839..5859 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 5875..6122 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6140..6159) /label=T7 /note="T7 promoter, forward primer" promoter complement(6141..6159) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6169..6186) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6169..6185) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6178..6200) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6326..6781 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6413..6432) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6623..6644 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6912..7715) /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNSCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" promoter complement(7716..7931) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(7955..7974) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8074..8096 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8134..8152) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 8193..9535 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 8525..8572 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 9562..9666 /gene="bla" /label=AmpR promoter CDS 9667..10527 /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(9885..9904) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 10698..180 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 61 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 121 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 181 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 241 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 301 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 361 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 421 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 481 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga 541 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc 601 gcaattaacc ctcactaaag ggaacaaaag ctggagctag tatactcttt cttcaacaat 661 taaatactct cggtagccaa gttggtttaa ggcgcaagac tgtaatttat cactacgaaa 721 tcttgagatc gggcgttcga ctcgcccccg ggagagatgg ccggcatggt cccagcctcc 781 tcgctggcgc cggctgggca acaccttcgg gtggcgaatg ggactttata cggtcagggt 841 agcgcccgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg 901 aaaaagtggc accgagtcgg tgcttttttt attttttgtc actattgtta tgtaaaatgc 961 cacctctgac agtatggaac gcaaacttct gtctagtgga tagtcgacat ccttttgttg 1021 tttccgggtg tacaatatgg acttcctctt ttctggcaac caaacccata catcgggatt 1081 cctataatac cttcgttggt ctccctaaca tgtaggtggc ggaggggaga tatacaatag 1141 aacagatacc agacaagaca taatgggcta aacaagacta caccaattac actgcctcat 1201 tgatggtggt acataacgaa ctaatactgt agccctagac ttgatagcca tcatcatatc 1261 gaagtttcac tacccttttt ccatttgcca tctattgaag taataatagg cgcatgcaac 1321 ttcttttctt tttttttctt ttctctctcc cccgttgttg tctcaccata tccgcaatga 1381 caaaaaaatg atggaagaca ctaaaggaaa aaattaacga caaagacagc accaacagat 1441 gtcgttgttc cagagctgat gaggggtatc tcgaagcaca cgaaactttt tccttccttc 1501 attcacgcac actactctct aatgagcaac ggtatacggc cttccttcca gttacttgaa 1561 tttgaaataa aaaaaagttt gctgtcttgc tatcaagtat aaatagacct gcaattatta 1621 atcttttgtt tcctcgtcat tgttctcgtt ccctttcttc cttgtttctt tttctgcaca 1681 atatttcaag ctataccaag catacaatca acttggaaaa aaatggacaa gaagtactcc 1741 attgggctcg atatcggcac aaacagcgtc ggttgggccg tcattacgga cgagtacaag 1801 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac 1861 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga 1921 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt 1981 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg 2041 gtggaggagg ataaaaagaa cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg 2101 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact 2161 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga 2221 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc 2281 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt 2341 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc 2401 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc 2461 gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg 2521 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac 2581 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg 2641 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat 2701 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag 2761 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga 2821 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc 2881 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac 2941 aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa 3001 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt 3061 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact 3121 cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc 3181 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag 3241 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag 3301 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa 3361 gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa 3421 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc 3481 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc 3541 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt 3601 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa 3661 gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg 3721 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat 3781 ggatttgcca accggaactt catgcagttg atccatgatg actctctcac ctttaaggag 3841 gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat 3901 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa 3961 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag 4021 aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag 4081 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt 4141 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag 4201 gaactggaca tcaatcggct ctccgactac gacgtggatc atatcgtgcc ccagtctttt 4261 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag 4321 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg 4381 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt 4441 ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag 4501 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat 4561 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc 4621 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat 4681 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct 4741 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag 4801 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc 4861 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac 4921 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc 4981 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc 5041 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg 5101 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg 5161 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc 5221 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga 5281 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt 5341 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg 5401 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa 5461 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt 5521 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac 5581 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca 5641 gaaaacatta tctacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac 5701 ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca 5761 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt 5821 ggagacagca gggctgaccc caagaagaag aggaaggtgt gatctcttct cgagtcatgt 5881 aattagttat gtcacgctta cgttcacgcc ctccccccac atccgctcta accgaaaagg 5941 aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat 6001 taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat 6061 gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt 6121 gcggccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 6181 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 6241 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 6301 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt 6361 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 6421 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 6481 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 6541 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 6601 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 6661 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 6721 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 6781 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagggtaat 6841 aactgatata attaaattga agctctaatt tgtgagttta gtatacatgc atttacttat 6901 aatacagttt tttagttttg ctggccgcat cttctcaaat atgcttccca gcctgctttt 6961 ctgtaacgtt caccctctac cttagcatcc cttccctttg caaatagtcc tcttccaaca 7021 ataataatgt cagatcctgt agagaccaca tcatccacgg ttctatactg ttgacccaat 7081 gcgtctccct tgtcatctaa acccacaccg ggtgtcataa tcaaccaatc gtaaccttca 7141 tctcttccac ccatgtctct ttgagcaata aagccgataa caaaatcttt gtcgctcttc 7201 gcaatgtcaa cagtaccctt agtatattct ccagtagata gggagccctt gcatgacaat 7261 tctgctaaca tcaaaaggcc tctaggttcc tttgttactt cttctgccgc ctgcttcaaa 7321 ccgctaacaa tacctgggcc caccacaccg tgtgcattcg taatgtctgc ccattctgct 7381 attctgtata cacccgcaga gtactgcaat ttgactgtat taccaatgtc agcaaatttt 7441 ctgtcttcga agagtaaaaa attgtacttg gcggataatg cctttagcgg cttaactgtg 7501 ccctccatgg aaaaatcagt caagatatcc acatgtgttt ttagtaaaca aattttggga 7561 cctaatgctt caactaactc cagtaattcc ttggtggtac gaacatccaa tgaagcacac 7621 gagtttgttt gcttttcgtg catgatatta aatagcttgg cagcaacagg actaggatga 7681 gtagcagcac gttccttata tgtagctttc gacatgattt atcttcgttt cctgcaggtt 7741 tttgttctgt gcagttgggt taagaatact gggcaatttc atgtttcttc aacactacat 7801 atgcgtatat ataccaatct aagtctgtgc tccttccttc gttcttcctt ctgttcggag 7861 attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat 7921 gatgaattga aaaggtggta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 7981 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 8041 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 8101 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 8161 ttaatgtcat gataataatg gtttcttagt atgatccaat atcaaaggaa atgatagcat 8221 tgaaggatga gactaatcca attgaggagt ggcagcatat agaacagcta aagggtagtg 8281 ctgaaggaag catacgatac cccgcatgga atgggataat atcacaggag gtactagact 8341 acctttcatc ctacataaat agacgcatat aagtacgcat ttaagcataa acacgcacta 8401 tgccgttctt ctcatgtata tatatataca ggcaacacgc agatataggt gcgacgtgaa 8461 cagtgagctg tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg 8521 ctttgaagtt cctattccga agttcctatt ctctagaaag tataggaact tcagagcgct 8581 tttgaaaacc aaaagcgctc tgaagacgca ctttcaaaaa accaaaaacg caccggactg 8641 taacgagcta ctaaaatatt gcgaataccg cttccacaaa cattgctcaa aagtatctct 8701 ttgctatata tctctgtgct atatccctat ataacctacc catccacctt tcgctccttg 8761 aacttgcatc taaactcgac ctctacattt tttatgttta tctctagtat tactctttag 8821 acaaaaaaat tgtagtaaga actattcata gagtgaatcg aaaacaatac gaaaatgtaa 8881 acatttccta tacgtagtat atagagacaa aatagaagaa accgttcata attttctgac 8941 caatgaagaa tcatcaacgc tatcactttc tgttcacaaa gtatgcgcaa tccacatcgg 9001 tatagaatat aatcggggat gcctttatct tgaaaaaatg cacccgcagc ttcgctagta 9061 atcagtaaac gcgggaagtg gagtcaggct ttttttatgg aagagaaaat agacaccaaa 9121 gtagccttct tctaacctta acggacctac agtgcaaaaa gttatcaaga gactgcatta 9181 tagagcgcac aaaggagaaa aaaagtaatc taagatgctt tgttagaaaa atagcgctct 9241 cgggatgcat ttttgtagaa caaaaaagaa gtatagattc tttgttggta aaatagcgct 9301 ctcgcgttgc atttctgttc tgtaaaaatg cagctcagat tctttgtttg aaaaattagc 9361 gctctcgcgt tgcatttttg ttttacaaaa atgaagcaca gattcttcgt tggtaaaata 9421 gcgctttcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa 9481 ttagcgctct cgcgttgcat ttttgttcta caaaatgaag cacagatgct tcgttcaggt 9541 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 9601 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 9661 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 9721 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 9781 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 9841 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 9901 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 9961 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 10021 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 10081 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 10141 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 10201 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 10261 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 10321 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 10381 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 10441 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 10501 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 10561 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 10621 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 10681 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 10741 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 10801 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 10861 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 10921 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 10981 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 11041 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 11101 gccacg //