LOCUS Exported 15440 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of both nmt41p-cas9 and rrk1p-gRNA (LEU2 marker) for CRISPR genome editing in fission yeast. ACCESSION . VERSION . KEYWORDS pAH237 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15440) AUTHORS Hayashi A, Tanaka K TITLE Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast. JOURNAL G3 (Bethesda). 2019 Feb 12. pii: g3.118.200976. doi: 10.1534/g3.118.200976. PUBMED 30755408 REFERENCE 2 (bases 1 to 15440) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15440 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS complement(722..1816) /codon_start=1 /gene="S. cerevisiae LEU2" /product="3-isopropylmalate dehydrogenase, required for leucine biosynthesis" /label=LEU2 /note="yeast auxotrophic marker" /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG DLGGSNSTTEVGDAVAEEVKKILA" promoter complement(1817..2224) /gene="S. cerevisiae LEU2" /label=LEU2 promoter promoter 3437..3793 /label=rrk1 promoter/leader /note="promoter and leader RNA for the Schizosaccharomyces pombe rrk1 gene encoding the K-RNA component of RNase P (Jacobs et al., 2014)" misc_RNA 3900..3951 /label=sTRSV HHRz /note="hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)" promoter 4857..6016 /label=nmt1 P41 promoter /note="mutant nmt1 promoter from Schizosaccharomyces pombe, conferring medium strength thiamine-repressible expression" CDS 6024..6089 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 6096..6116 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6141..10241 /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 10242..10289 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" rep_origin 11436..12219 /label=ars1 /note="Schizosaccharomyces pombe autonomously replicating sequence ars1" primer_bind complement(12569..12586) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(12569..12585) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12578..12600) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 12798..13253 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(12885..12904) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 13095..13116 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(13270..13289) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 13389..13411 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(13449..13467) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 13535..13639 /gene="bla" /label=AmpR promoter CDS 13640..14500 /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(13858..13877) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 14671..15259 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 15160..15179 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 15413..15430 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 241 tcgatcgact acgtcgttaa ggccgtttct gacagagtaa aattcttgag ggaactttca 301 ccattatggg aaatggttca agaaggtatt gacttaaact ccatcaaatg gtcaggtcat 361 tgagtgtttt ttatttgttg tatttttttt tttttagaga aaatcctcca atatataaat 421 taggaatcat agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc 481 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc 541 aatttgctta cctgtattcc tttacatcct cctttttctc cttcttgata aatgtatgta 601 gattgcgtat atagtttcgt ctaccctatg aacatattcc attttgtaat ttcgtgtcgt 661 ttctattatg aatttcattt ataaagttta tgtacaaata tcataaaaaa agagaatctt 721 tttaagcaag gattttctta acttcttcgg cgacagcatc accgacttcc gtggtactgt 781 tggaaccacc taaatcacca gttctgatac ctgcatccaa aaccttttta actgcatctt 841 caatggcctt accttcttca ggcaagttca atgacaattt caacatcatt gcagcagaca 901 agatagtggc gatagggttg accttattct ttggcaaatc tggagcagaa ccgtggcatg 961 gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac gcagatggca 1021 acaaacccaa ggaacctggg ataacggagg cttcatcgga gatgatatca ccaaacatgt 1081 tgctggtgat tataatacca tttaggtggg ttgggttctt aactaggatc atggcggcag 1141 aatcaatcaa ttgatgttga accttcaatg tagggaattc gttcttgatg gtttcctcca 1201 cagtttttct ccataatctt gaagaggcca aaacattagc tttatccaag gaccaaatag 1261 gcaatggtgg ctcatgttgt agggccatga aagcggccat tcttgtgatt ctttgcactt 1321 ctggaacggt gtattgttca ctatcccaag cgacaccatc accatcatct tcctttctct 1381 taccaaagta aatacctccc actaattctc tgacaacaac gaagtcagta cctttagcaa 1441 attgtggctt gattggagat aagtctaaaa gagagtcgga tgcaaagtta catggtctta 1501 agttggcgta caattgaagt tctttacgga tttttagtaa accttgttca ggtctaacac 1561 taccggtacc ccatttagga ccacccacag cacctaacaa aacggcatca accttcttgg 1621 aggcttccag cgcctcatct ggaagtggga cacctgtagc atcgatagca gcaccaccaa 1681 ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac atcagaaata gctttaagaa 1741 ccttaatggc ttcggctgtg atttcttgac caacgtggtc acctggcaaa acgacgatct 1801 tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat attgctgaaa 1861 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca 1921 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa 1981 cgcttctcta ttctatatga aaagccggtt ccgcggctct cacctttcct ttttctccca 2041 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc 2101 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa 2161 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac 2221 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac 2281 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac 2341 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt 2401 tgggattcca tttttaataa ggcaataata ttaggtatgt agatatacta gaagttctcc 2461 tcgacaagct tgcatgcctg cagggaaatc ataacatagt ataataggaa tcatatgaca 2521 tttactcttg catagcaata tcctcttctt cgccatcagc aatccgacga gcatatgctt 2581 cagcctcttg caaaagctca tcaggaacgg gtgtatgaat accgtttgtt tcaacaccaa 2641 cccaagtcat ctcaatttca tgctctttgt tgttttcttc gtcatgagtt gcgtataatc 2701 taaaaaagag tgaattatta gcaaatagaa tgaaaaaaaa ttgtactcaa caaagcaaaa 2761 cgtacattct agcagcttct ttaaccgcat ctttcatttt taatgaggaa aaattcagtt 2821 tttctaattc gtttcgagct acttgccgac ctttaccagc tgcagcaccg ttgtaacccc 2881 aataaacacc attaggttca accatataca aatgtggacc ttctgagtca taagtagcaa 2941 caaaagacat aacaccgaat ggacggacgc tagaatagca agtgaaaagt tgaacgtagt 3001 tccccaagcg atcagcaata actgttccag ggatgggaga tccgtagtta tctctccatg 3061 aggtggcttc gtcccttgca cgttttacga tgtgttggcc atcaggaata aagccagtag 3121 tagcaatacc aatgtgtcga tcgaccgacc caattctgtt atttactcgc ggcttaagta 3181 gtttgctagt aactaccttt tctagagcta gaataactcc atcttcacat tttattccaa 3241 tgcaagtact aaatacaatg ttaaaatcaa tcgataagga aattcaatcc accttacctt 3301 gcattttcga cggctttata cgcgtactcc gcttggaata aacgaccatc cggggagaag 3361 aataatccta aatcgtatcc agtaccaatt gaactctgtt tgttaatgtg atgtgctact 3421 ccttgctaac ttaccatttg cttatgttgg tggtagttgg catgcgtaga ctgatgacta 3481 gtcagcaagg agcgtagaac agtcacactc gttatatatg tgcttccaag aaaactcaag 3541 aatttaccat tagcaaacac ttttttgaaa tgttagacat ttaaatgacg aaggcatata 3601 gaagctttga ataggtgttg taaagtgttg atttatgtga cgctgagggt gcgcatgaaa 3661 ggaatgttgg gtcacgatta ttaaacagtt tgctagcttg gacacttgag tattggaagt 3721 tgttgaattc taaaaaactt tcagttgatt tgaatagttg ctgttgccaa aaaacataac 3781 ctgtaccaaa gaacaccggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt 3841 aaaataaggc tagtccgtta tcaacttgaa aaaagtgagt ggcaccgagt cggtggtgcc 3901 ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag gtacggttat 3961 ttgttagcaa tggtaggaga aaattaggga ttgtttacca aaacaacgtg aacaaatagt 4021 tatagtttta atttaactca tgaacatatt atcggaaaaa atctactcat ttatgtctgg 4081 gtttagctcc ctttatggtt tcgttttgca gtagaataca ctatggtatt tattttgtgc 4141 actacaaacc gtgcttttta tgtgaacgtt ttgatgaaaa cgttctagag attgatttat 4201 tattcaatgt taaagtaact cattttgcta tatgtatctt tttttcagcg aactttcgtt 4261 actgagcagt tcaagcagtg atttggcatt atggtatcaa taaagacata tcattaaggc 4321 aaggctacgg aaagaaaaaa gaaggttttt aaattaatat acaacaaatt aaccgtgatc 4381 ggacagtata tttgatgcaa agatataatg aaagttggga aaataagaat atagcactca 4441 aatgattgcc ttacaaaagc attgctttct ctacaggtaa ggttgaagtg cctttacggt 4501 atcttcaaaa accgcatcgg gatcttgctc ggcatcgatg gttatcagac gattttgtga 4561 tttcagatat tcgacgacag gcatgctggc tttagtatat gtaacaaagc gtttcttgat 4621 cgactcaatg ttgtcatcag aacggccact agtcttacca cgatgaataa gacgcttcaa 4681 catggtttcc tgaccacaac ggaaataaag agcaaactta gcggggcaga ccgacttttc 4741 aaaaccttcg cattgatcca tctcacgggg gaaaccgtca atcaagaact tgtcaattcc 4801 tttatcatgg cattccttca ttttagtctc taaaagggat atagttattt ccatggtcgc 4861 cataaaagac agaataagtc atcagcggtt gtttcatttc ctatattttt tttttatttt 4921 tttatttttt aataagggaa aatttaacgt ctaaggatac agaagattgt tagcacatta 4981 aagtaataaa ggcttaagta gtaagtgcct tagcatgtta ttgtatttca aaggacataa 5041 tctaaaataa taacaatatc atttctcaca agttattcaa ttttcttttt tttttctaat 5101 aatatcaaga atgtattatt tgtttgacat aagtcaacta atttatttaa tatgctggat 5161 taatcttgca gacatgtaaa ttaacaagtt ttagtcaaat aacgttgaag tttcaatgaa 5221 ctcaaataat ttctcttttt ttttatataa ccataagtct aatctgattt atattttccg 5281 cagggatcaa ctgaagttat gacatttgga ttggatcact tataaccttg gtcgccaaat 5341 aatacaaaaa tcagcgttat aaaacaaaga aggtttttgt taagaaatta atcctctttc 5401 ttgataagaa agttgaaccg aaattgcaga tactgatata tgaaaataat acccacaatt 5461 ttgggaatag cgcaagcctc aatttaaaca ataggtgagg acacatgata atgacctcaa 5521 tgattgttag aagaaaagag cctcattaca aaatcgaaaa atgaatggtt gggtacaagt 5581 ttccaaaaca tggtaaagtg gactttgcgt atgagacgta aatagaaaaa aacacttgtt 5641 atatgttttc tagaattatt gttgtctctt tatggttgga tgatgcaaaa tagtaatttc 5701 ggttagttgc tgtaaaacac cacgagacaa atagatatgg atatttatta aatcaggaaa 5761 aacgtaactc tcggctactg gatggttcag tcacccaacg attactgggg agagaaaaca 5821 gggcaaaagc aaagcttaaa ggaatccgat tgtcattcgg caatgtgcag cgaaactaaa 5881 aaccggataa tggacctgtt aatcgaaaca ttgaagataa aggaagagga atcctggcat 5941 atcatcaatt gaataagttg aattaattat ttcaatctca ttctcacttt ctgacttata 6001 gtcgctttgt taaatctcga atggactata aggaccacga cggagactac aaggatcatg 6061 atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta 6121 tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact 6181 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc 6241 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca 6301 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac 6361 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg 6421 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc 6481 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca 6541 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct 6601 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga 6661 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc 6721 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca 6781 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga 6841 atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca 6901 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc 6961 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga 7021 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg 7081 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc 7141 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga 7201 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt 7261 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca 7321 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc 7381 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg 7441 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc 7501 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc 7561 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga 7621 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt 7681 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga 7741 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga 7801 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct 7861 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc 7921 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca 7981 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac 8041 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga 8101 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt 8161 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc 8221 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg 8281 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga 8341 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca 8401 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga 8461 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga 8521 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact 8581 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg 8641 actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca 8701 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg 8761 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga 8821 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg 8881 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc 8941 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag 9001 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag 9061 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa 9121 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg 9181 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca 9241 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg 9301 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg 9361 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg 9421 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga 9481 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg 9541 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca 9601 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg 9661 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga 9721 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg 9781 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact 9841 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga 9901 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg 9961 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca 10021 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc 10081 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga 10141 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc 10201 tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga 10261 aaaaggccgg ccaggcaaaa aagaaaaagt aagatctccc gggtaaaagg aatgtctccc 10321 ttgccagtac tgctagggtt tttctttcaa actatggaag cccattcaag ctgcatatta 10381 cgattttgtt tttcgctttt agaaagtggt ttagatgaga taatagaaaa attcttgatc 10441 tccgacaacg agtactttta ttttttttgc taatcacttt actcaatatt agctcgaaat 10501 cgtagaaacg tagacgggtg cgggataccg agtggtgtag ttaagaattt ttataaacca 10561 cgtggcccaa aaatatgaac ccaaaacgtt tatacatgag tatactttaa gaaggctata 10621 ccccttcgtg ttagatgtag ttttagctac ccaacccgag tctatgagct tgacttcaga 10681 tgtagaaggc attaaatcgt tttgaatatt aattaaaaaa cgatgaaaat taaatattta 10741 aaagcaatca tacgctgaaa atttagtgct gtggctaatc cttcaacatg gaaatgccat 10801 aaaagtgact ttgacaaaaa aaaaagtata tacaggtagt aaactcatct acttcattga 10861 ctttgtttac agcatgtgga aggaggaata tttattgcta aatcgtagtt taacattcaa 10921 taagtaatac tattgaaatt cgacaagatt ggccgcatgg atgaaaaaga ggcattttgc 10981 tttgggagaa ttagttcaaa ttagaactga aaaaaaaaac tttacgaggc aaaaatgtcg 11041 gattgagatc gtaaaagttc gctcgtcgtc ttttgctttg tgattgtttt catggataca 11101 tcttgctgga tatttaaatt ttagtactat gtataagata ttctataaat gttttatcac 11161 ccaaacctgt tagcgccttc ttaattctat tcaatctggc ttttgctctg agactacttc 11221 ttggactttc actacttgtt agttatacgg aatttgtgta attagaagtg aaataatcct 11281 ttctattagt aatgccatgg gcacaatttt accatctttg atatattctt taataaggtt 11341 tccgtatttg ctgccaggac gcgaattcga gtctaactcc ttaaccactc ggacatagtg 11401 acttatctga cactcattgt aaattaatat atatataggc attttgttta gttaaaggta 11461 cttaagtaat tagtataaac gaaccaattt tataatcagg aagttaagtg aatggtagca 11521 catgtcgtaa aaattgtgaa tttttattga ataatatttt aaatacaagc ctttctagac 11581 taggtatact cataaacata tatgagcaaa aggatagagg agattacatt gcatcttcta 11641 caaatttatt tattgccctt tactgaaaaa ttaaataatg agtactaaat gataaaaagc 11701 gctcagtaca agaaagattg caaaaatatt gcattcttca tgaattaaag ttgcatataa 11761 ggcatattga aagtaatagt actaaaacag cagttagcga aaattaatag aattatattc 11821 gcaagacaat tgtgacatta aattaaaaaa ttgtagaatt tttactatcc tctttaacgc 11881 catgagcctt tataaaaagg ttaaattagt tttaacattc tttttttgag taagagttta 11941 caatttatca aaacctgtgt tattatattc attagtttca atttattagc atctagagaa 12001 aaatcaattg gcagttacat tgttggaatt tatgaagaaa aagaatctac aacggagaat 12061 aagttgctga tcgctttccc taaaattgta tattttgctg agcttatttt gacatttcgt 12121 tgaagttttc tctaattcgc attcatttta agtaaaacaa tgagaaataa aattacaaaa 12181 aatacaaatt aaaatacaat ttttagctat aattatagac gatgcccttg tatcccattc 12241 tgtctcgctt gcccctactt tttatctttt atataccata atgaacgctg ccgctactaa 12301 ccataccccg attttacatt tcggactccc aaggacgtac aaaatagaaa actatagaaa 12361 aaaataatca gaaaatagca tgtcatctct ttgtaaaacg cgtttgcaag aagaaaggaa 12421 acaatggaga agagatcatc catttgtatg taaattttag taaacttgaa gaaatcacta 12481 acaacttctc ttacttaggg attctatgca aaaccttgta aatcatctga tggaggactc 12541 gatttaatga attggaaggt tggaattcac tggccgtcgt tttacaacgt cgtgactggg 12601 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 12661 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 12721 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac 12781 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 12841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 12901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 12961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga 13021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 13081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg 13141 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 13201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg 13261 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 13321 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 13381 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag 13441 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 13501 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 13561 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 13621 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 13681 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 13741 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 13801 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 13861 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 13921 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 13981 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 14041 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 14101 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 14161 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 14221 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 14281 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 14341 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 14401 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 14461 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 14521 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 14581 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 14641 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 14701 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 14761 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 14821 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 14881 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 14941 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 15001 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 15061 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 15121 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 15181 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 15241 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 15301 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 15361 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 15421 agtgagcgag gaagcggaag //