LOCUS Exported 11667 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION A CRISPR activation device with the necessary genes (dxcas9 controlled by Ptet, pspF-Delta-HTH::lambda-N22plus controlled by promoter PrhaB), and the reporter part (PpspA-LEA3B3 with sfgfp::ASV).. ACCESSION . VERSION . KEYWORDS pLY70 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11667) AUTHORS TITLE Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria JOURNAL Nature Communications 10, August 2019 REFERENCE 2 (bases 1 to 11667) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11667 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(166..1116) /codon_start=1 /gene="rep101" /product="RepA protein needed for replication with the pSC101 origin" /label=Rep101 /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 1164..1386 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" terminator 1878..1972 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(2002..2862) /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 2625..2644 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2863..2967) /gene="bla" /label=AmpR promoter primer_bind 3032..3050 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 3054..3097 /label=bacterial terminator /note="putative bacterial transcription terminator" misc_feature 3100..3121 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" CDS complement(3188..7291) /codon_start=1 /product="catalytically dead Streptococcus pyogenes Cas9 mutant with broad PAM compatibility (Hu et al., 2018)" /label=dxCas9(3.7) /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" RBS 7298..7309 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" promoter 7327..7382 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 7328..7346 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 7401..8024 /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(7474..7493) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" CDS 8039..8050 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS complement(8194..9030) /codon_start=1 /gene="E. coli rhaS" /product="positive regulator of the rhaB promoter " /label=rhaS /note="activated by L-rhamnose" /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ" terminator 9098..9125 /label=T7Te terminator /note="phage T7 early transcription terminator" promoter 9183..9301 /label=rhaB promoter /note="promoter of the E. coli rhaBAD operon, conferring tight induction with L-rhamnose and repression with D-glucose in the presence of RhaR and RhaS (Giacalone et al., 2006)" CDS 10299..10310 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" misc_RNA 10532..10581 /label=sTRSV HHRz /note="hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)" RBS 10614..10625 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 10632..11345 /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" misc_feature 11452..11472 /label=BioBrick suffix /note="universal suffix for all parts" terminator 11473..11530 /label=his operon terminator /note="This putative transcriptin terminator from the E. coli his operon has a 2-bp deletion introduced during synthesis. Its efficiency has not been determined." primer_bind complement(11554..11571) /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 aatgcaccaa aaactcgtaa aagctctgat gtatctatct tttttacacc gttttcatct 61 gtgcatatgg acagttttcc ctttgatatg taacggtgaa cagttgttct acttttgttt 121 gttagtcttg atgcttcact gatagataca agagccataa gaacctcaga tccttccgta 181 tttagccagt atgttctcta gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc 241 attgagatca tacttacttt gcatgtcact caaaaatttt gcctcaaaac tggtgagctg 301 aatttttgca gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc 361 tgatgtaatg gttgttggta ttttgtcacc attcattttt atctggttgt tctcaagttc 421 ggttacgaga tccatttgtc tatctagttc aacttggaaa atcaacgtat cagtcgggcg 481 gcctcgctta tcaaccacca atttcatatt gctgtaagtg tttaaatctt tacttattgg 541 tttcaaaacc cattggttaa gccttttaaa ctcatggtag ttattttcaa gcattaacat 601 gaacttaaat tcatcaaggc taatctctat atttgccttg tgagttttct tttgtgttag 661 ttcttttaat aaccactcat aaatcctcat agagtatttg ttttcaaaag acttaacatg 721 ttccagatta tattttatga atttttttaa ctggaaaaga taaggcaata tctcttcact 781 aaaaactaat tctaattttt cgcttgagaa cttggcatag tttgtccact ggaaaatctc 841 aaagccttta accaaaggat tcctgatttc cacagttctc gtcatcagct ctctggttgc 901 tttagctaat acaccataag cattttccct actgatgttc atcatctgag cgtattggtt 961 ataagtgaac gataccgtcc gttctttcct tgtagggttt tcaatcgtgg ggttgagtag 1021 tgccacacag cataaaatta gcttggtttc atgctccgtt aagtcatagc gactaatcgc 1081 tagttcattt gctttgaaaa caactaattc agacatacat ctcaattggt ctaggtgatt 1141 ttaatcacta taccaattga gatgggctag tcaatgataa ttacatgtcc ttttcctttg 1201 agttgtgggt atctgtaaat tctgctagac ctttgctgga aaacttgtaa attctgctag 1261 accctctgta aattccgcta gacctttgtg tgtttttttt gtttatattc aagtggttat 1321 aatttataga ataaagaaag aataaaaaaa gataaaaaga atagatccca gccctgtgta 1381 taactcacta ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa cgctgtttgc 1441 tcctctacaa aacagacctt aaaaccctaa aggcttaagt agcaccctcg caagctcggg 1501 caaatcgctg aatattcctt ttgtctccga ccatcaggca cctgagtcgc tgtctttttc 1561 gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa tggcactaca 1621 ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag cccgtcacgg 1681 gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc tatctgactt tttgctgttc 1741 agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg tgacaggtca 1801 ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacaggctt acccgtctta 1861 ctgtccctag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg 1921 aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag tccaagcgag 1981 ctcgtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 2041 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 2101 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 2161 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 2221 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 2281 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 2341 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 2401 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 2461 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 2521 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 2581 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 2641 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 2701 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 2761 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 2821 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 2881 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 2941 gaaaaataaa caaatagggg ttccgcgcac atttcccatg gtgccacctg acgtctaaga 3001 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc agaatttcag 3061 ataaaaaaaa tccttagctt tcgctaagga tgatttctgg aattcgcggc cgcttctaga 3121 ggggagacca gaaacaaaaa aaggggagcg gtttcccgct ccccttcaat aattggctct 3181 agtattagtc acctcctagc tgactcaaat caatgcgtgt ttcataaaga ccagtgatgg 3241 attgatggat aagagtggca tctaaaactt cttttgtaga cgtatatcgt ttacgatcaa 3301 ttgttgtatc aaaatattta aaagcagcgg gagctccaag attcgtcaac gtaaataaat 3361 gaataatatt ttctgcttgt tcacgtattg gtttgtctct atgtttgtta tatgcactaa 3421 gaactttatc taaattggca tctgctaaaa taacacgctt agaaaattca ctgatttgct 3481 caataatctc atctaaataa tgcttatgct gctccacaaa caattgtttt tgttcgttat 3541 cttctggact acccttcaac ttttcataat gactagctaa atataaaaaa ttcacatatt 3601 tgcttggcag agccagctca tttccttttt gtaaaactcc ggcactagcc agcatccgtt 3661 tacgaccgtt ttctaactca aaaagactat atttaggtag tttaatgatt aagtcttttt 3721 taacttcctt atatccttta gcttctaaaa agtcaatcgg atttttttca aaggaacttc 3781 tttccataat tgtgatccct agtaactctt taacggattt taacttcttc gatttccctt 3841 tttccacctt agcaaccact aggactgaat aagctaccgt tggactatca aaaccaccat 3901 atttttttgg atcccagtct tttttacgag caataagctt gtccgaattt ctttttggta 3961 aaattgactc cttggagaat ccgcctgtct gtacttctgt tttcttgaca atattgactt 4021 ggggcatgga caatactttg cgcactgtgg caaaatctcg ccctttatcc cagacaattt 4081 ctccagtttc cccattagtt tcgattagag ggcgtttgcg aatctctcca tttgcaagtg 4141 taatttctgt tttgaagaag ttcatgatat tagagtaaaa gaaatatttt gcggttgctt 4201 tgcctatttc ttgctcagac ttagcaatca ttttacgaac atcataaact ttataatcac 4261 catagacaaa ctccgattca agttttggat atttcttaat caaagcagtt ccaacgacgg 4321 catttagata cgcatcatgg gcatgatggt aattgttaat ctcacgtact ttatagaatt 4381 ggaaatcttt tcggaagtca gaaactaatt tagattttaa ggtaatcact ttaacctctc 4441 gaataagttt atcattttca tcgtatttag tattcatgcg actatccaaa atttgtgcca 4501 catgcttagt gatttggcga gtttcaacca attggcgttt gataaaacca gctttatcaa 4561 gttcactcaa acctccacgt tcagctttcg ttaaattatc aaacttacgt tgagtgatta 4621 acttggcgtt tagaagttgt ctccaatagt ttttcatctt tttgactact tcttcacttg 4681 gaacgttatc cgatttacca cgatttttat cagaacgcgt taagacctta ttgtctattg 4741 aatcgtcttt aaggaaactt tgtggaacaa tggcatcgac atcataatca cttaaacgat 4801 taatatctaa ttcttggtcc acatacatgt ctcttccatt ttggagataa tagagataga 4861 gcttttcatt ttgcaattga gtattttcaa caggatgctc tttaagaatc tgacttccta 4921 attctttgat accttcttcg attcgtttca tacgctctcg cgaatttttc tggccctttt 4981 gagttgtctg attttcacgt gccatttcaa taacgatatt ttctggctta tgccgcccca 5041 ttactttgac caattcatca acaactttta cagtctgtaa aatacctttt ttaatagcag 5101 ggctaccagc taaatttgca atatgttcat gtaaactatc gccttgtcca gacacttgtg 5161 ctttttgaat gtcttcttta aatgtcaaac tatcatcatg gatcagctga ataaaattgc 5221 gattggcaaa accatctgat ttcaaaaaat ctaatattgt tttgccagat tgcttatccc 5281 taataccatt aatcaatttt cgagacaaac gtccccaacc agtataacgg cgacgtttaa 5341 gctgtttcat caccttatca tcaaagaggt gagcatatgt tttaagtctt tcctcaatca 5401 tctccctatc ttcaaataag gtcaatgtta aaacaatatc ctctaagata tcttcatttt 5461 cttcattatc caaaaaatct ttatctttaa taatttttag caaatcatgg taggtaccta 5521 atgaagcatt aaatctatct tcaactcctg aaatttcaac actatcaaaa cattctattt 5581 ttttgaaata atcttctttt aattgcttaa cggttacttt tcgatttgtt ttgaagagta 5641 aatcaacaat ggctttcttc tgatcacctg aaagaaatgc tggttttcgc attccttcag 5701 taacatattt gacctttgtc aattcgttat aaaccgtaaa atactcataa agcaaactat 5761 gttttggtag tactttttca tttggaagat ttttatcaaa gtttgtcatg cgttcaataa 5821 atgattgagc tgaagcacct ttatcgacaa ctttttcaaa attccatggg gtaattgttt 5881 cttcagactt ccgagtcatc catgcaaaac gactattgcc acgcgccaat ggaccaacat 5941 aataaggaat acgaaaagtc aagatttttt caatcttctc acgattgtct tttaaaaatg 6001 gataaaagtc ttcttgtctt ctcaaaatag catgcagctc acccaagtga atttgatggg 6061 gaataatgcc gttgtcaaag gtccgttgct tgcgcagcaa atcttcacga tttagtttca 6121 ccaataattc ctcagtacca tccatttttt ctaaaattgg tttgataaat ttataaaatt 6181 cttcttggct agctccccca tcaatataac ctgcatatcc gttttttgat tgatcaaaaa 6241 agatttcttt atacttttct ggaagttgtt gtcgaactaa agcttttaaa agagtcaagt 6301 cttgatgatg ttcatcgtat aatttaatca ttgaagctga taggggagcc ttagttattt 6361 cagtatttac tcttaggata tctgaaagta aaatagcatc tgataaattc ttagctgcca 6421 aaaacaaatc agcatattga tctccaattt gcgccaataa attatctaaa tcatcatcgt 6481 aagtatcttt tgaaagctgt aatttggtat cttctgccaa atcaaaattt gatttaaaat 6541 taggggtcaa acccaatgac aaagcaatga gattcccaaa taagccattt ttcttctcac 6601 cggggagctg agcaatgaga ttttctaatc gtcttgattt actcaatcgt gcagaaagaa 6661 tcgctttagc atctactcca cttgcgttaa tagggttttc ttcaaataat tgattgtagg 6721 tttgtaccaa ctggataaat agtttgtcca catcactatt atcaggattt aaatctccct 6781 caatcaaaaa atgaccacga aacttaatca tatgcgctaa ggccaaatag attaagcgca 6841 aatccgcttt atcagtagaa tctaccaatt tttttcgcag atgatagata gttggatatt 6901 tctcatgata agcaacttca tctactatat ttccaaaaat aggatgacgt tcatgcttct 6961 tgtcttcttc caccaaaaaa gactcttcaa gtcgatgaaa gaaactatca tctactttcg 7021 ccatctcatt tgaaaaaatc tcctgtagat aacaaatacg attcttccga cgtgtatacc 7081 ttctacgagc tgtccgtttg aggcgagtcg cttccgctgt ctctccactg tcaaataaaa 7141 gagcccctat aagatttttt ttgatactgt ggcggtctgt atttcccaga accttgaact 7201 ttttagacgg aaccttatat tcatcagtga tcaccgccca tccgacgcta tttgtgccga 7261 tagctaagcc tattgagtat ttcttatcca tagatccttt ctcctcttta gatcttttca 7321 attcttttct ctatcactga tagggagtgg taaaataact ctatcaacga tagagtgtca 7381 acaaaaatta ggaattaatg atgtcaagat tagataaaag taaagtgatt aacagcgcat 7441 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc 7501 taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg 7561 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa 7621 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc 7681 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg 7741 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac 7801 tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg 7861 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta 7921 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca 7981 tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ttaatactag agccaattat 8041 tgaaggccgc taacgcggcc tttttttgtt tctggtctcc ctactagagc ctagttgtct 8101 tcatgcatga agacaaaatt aatactagag ggaccaaaac gaaaaaagac gcttttcagc 8161 gtcttattgt tcgtctttgg taccgagcta gtattattgc agaaagccat cccgtccctg 8221 gcgaatatca cgcggtgacc agttaaactc tcggcgaaaa agcgtcgaaa agtggttact 8281 gtcgctgaat ccacagcgat aggcgatgtc agtaacgctg gcctcgctgt ggcgtagcag 8341 atgtcgggct ttcatcagtc gcaggcggtt caggtatcgc tgaggcgtca gtcccgtttg 8401 ctgcttaagc tgccgatgta gcgtacgcag tgaaagagaa aattgatccg ccacggcatc 8461 ccaattcacc tcatcggcaa aatggtcctc cagccaggcc agaagcaagt tgagacgtga 8521 tgcgctgttt tccaggttct cctgcaaact gcttttacgc agcaagagca gtaattgcat 8581 aaacaagatc tcgcgactgg cggtcgaggg taaatcattt tccccttcct gctgttccat 8641 ctgtgcaacc agctgtcgca cctgctgcaa tacgctgtgg ttaacgcgcc agtgagacgg 8701 atactgccca tccagctctt gtggcagcaa ctgattcagc ccggcgagaa actgaaatcg 8761 atccggcgag cgatacagca cattggtcag acacagatta tcggtatgtt catacagatg 8821 ccgatcatga tcgcgtacga aacagaccgt gccaccggtg atggtatagg gctgcccatt 8881 aaacacatga atacccgtgc catgttcgac aatcacaatt tcatgaaaat catgatgatg 8941 ttcaggaaaa tccgcctgcg ggagccgggg ttctatcgcc acggacgcgt taccggacgg 9001 aaaaaaatcc acactatgta atacggtcat ttgggctccc tctagtagct agcataatac 9061 ctaggactga gctagctgta aactctagta tcacactggc tcaccttcgg gtgggccttt 9121 ctgcgtttat atactagaga gacctttacg ccgctggagc aggaatgcgg tgagcatcac 9181 atcaccacaa ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaatg 9241 gcccattttc ctgtcagtaa cgagaaggtc gcgaattgag gcgcttttta gactggtcgt 9301 aactagagtc acacaggaaa gtactagatg gcagaataca aagataattt acttggtgag 9361 gcgaacagct ttctcgaagt gctggaacag gtttcgcatc tcgcaccgct ggacaaaccg 9421 gtgctcatca tcggcgaacg cggcaccggt aaagagctga ttgccagccg cctgcattat 9481 ctctcctccc gttggcaagg gccgtttatt tcccttaact gcgcggcgtt aaatgaaaat 9541 ctgctggatt ccgaactgtt tggtcacgaa gcgggggcgt ttaccggtgc gcaaaaacgt 9601 catccaggga gatttgaacg tgccgacggc ggtacgctat ttcttgatga actcgctacg 9661 gcaccgatga tggtgcagga gaaattattg cgcgtgattg agtacggtga actggagcgc 9721 gttggcggca gccaaccatt gcaggtgaat gtgcggttgg tatgcgcgac gaatgccgat 9781 ctcccggcga tggtcaatga aggcactttt cgcgctgacc tgctcgaccg actggctttt 9841 gatgttgtac aactgccacc actgcgcgag cgcgaaagcg acataatgtt gatggcagaa 9901 tactttgcca tccagatgtg tcgggaaatc aagctgcctc tgttcccggg gtttacggag 9961 cgcgccagag aaacattgct gaattatcgt tggccgggaa atattcgtga attgaaaaac 10021 gtggtggaac gttcagtgta tcgccacggc accagcgatt atccgcttga tgacatcatt 10081 attgatccct ttaaacggcg tccgcctgaa gacgctatcg ccgtttcaga aaccacctcg 10141 cttccaacac tgccgctgga tttacgtgag tttcagatgc agcaggaaaa agagttgctg 10201 caactcagtt tgcaaatgaa tgcacgcaca cgccgccgcg aacgtcgcgc agagaaacag 10261 gctcaatgga aagcagcaaa ttaatactag agccaattat tgaaggccgc taacgcggcc 10321 tttttttgtt tctggtctcc ctactagaag ttatttccag tgatcgcgcg gcatagttcg 10381 tttcccatcg gttagtgtaa ttcgctaact catcctggca tgttgctgtt gattcttcaa 10441 tcagatcttt ataaatcaaa aagataaaaa attggcacgc aaattgtatt aacagttcag 10501 caggacaatc ctgaacgcaa atactagaga gctgtcaccg gatgtgcttt ccggtctgat 10561 gagtccgtga ggacgaaaca gcctctacaa ataattttgt ttaactagag attaaagagg 10621 agaaatacca tatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct attctggtgg 10681 aactggatgg tgatgtcaac ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg 10741 caactaatgg taaactgacg ctgaagttca tctgtactac tggtaaactg ccggtacctt 10801 ggccgactct ggtaacgacg ctgacttatg gtgttcagtg ctttgctcgt tatccggacc 10861 atatgaagca gcatgacttc ttcaagtccg ccatgccgga aggctatgtg caggaacgca 10921 cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg 10981 ataccctggt aaaccgcatt gagctgaaag gcattgactt taaagaagac ggcaatatcc 11041 tgggccataa gctggaatac aattttaaca gccacaatgt ttacatcacc gccgataaac 11101 aaaaaaatgg cattaaagcg aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc 11161 agctggctga tcactaccag caaaacactc caatcggtga tggtcctgtt ctgctgccag 11221 acaatcacta tctgagcacg caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc 11281 atatggttct gctggagttc gtaaccgcag cgggcatcac gcatggtatg gatgaactgt 11341 acaaaaggcc tgctgcaaac gacgaaaact acgctgcatc agtttaataa tactagagcc 11401 aattattgaa ggcctcccta acggggggcc tttttttgtt tctggtctcc ctactagtag 11461 cggccgctgc agtccggcaa aaaagggcaa ggtgtcacca ccctgccctt tttctttaaa 11521 accgaaaaga ttacttcgcg ttatgcaggc ttcctcgctc actgactcgc tgcgctcggt 11581 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatctcga ggttacattg 11641 tcgatctgtt catggtgaac agctttg //