LOCUS Exported 10491 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION A CRISPR activation device with the necessary genes (dcas9 controlled by Ptet, pspF-Delta-HTH::lambda-N22plus controlled by promoter J23106), and the reporter part (PpspA-LEA1B1 with sfgfp::ASV).. ACCESSION . VERSION . KEYWORDS pLY53 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10491) AUTHORS TITLE Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria JOURNAL Nature Communications 10, August 2019 REFERENCE 2 (bases 1 to 10491) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10491 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 11..29 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 33..76 /label=bacterial terminator /note="putative bacterial transcription terminator" misc_feature 79..100 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" CDS complement(164..4270) /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" RBS 4277..4288 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" promoter 4306..4361 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 4307..4325 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 4380..5003 /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(4453..4472) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" CDS 5018..5029 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS 6102..6113 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" misc_RNA 6335..6384 /label=sTRSV HHRz /note="hammerhead ribozyme from the tobacco ringspot virus satellite RNA (Khvorova et al., 2003)" RBS 6417..6428 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 6435..7148 /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 7255..7275 /label=BioBrick suffix /note="universal suffix for all parts" terminator 7276..7333 /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(7357..7374) /label=L4440 /note="L4440 vector, forward primer" CDS complement(7636..8586) /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 8634..8856 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" terminator 9348..9442 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(9472..10332) /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 10095..10114 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10333..10437) /gene="bla" /label=AmpR promoter ORIGIN 1 aacctataaa aataggcgta tcacgaggca gaatttcaga taaaaaaaat ccttagcttt 61 cgctaaggat gatttctgga attcgcggcc gcttctagag gggagaccag aaacaaaaaa 121 aggggagcgg tttcccgctc cccttcaata attggctcta gtattagtca cctcctagct 181 gactcaaatc aatgcgtgtt tcataaagac cagtgatgga ttgatggata agagtggcat 241 ctaaaacttc ttttgtagac gtatatcgtt tacgatcaat tgttgtatca aaatatttaa 301 aagcagcggg agctccaaga ttcgtcaacg taaataaatg aataatattt tctgcttgtt 361 cacgtattgg tttgtctcta tgtttgttat atgcactaag aactttatct aaattggcat 421 ctgctaaaat aacacgctta gaaaattcac tgatttgctc aataatctca tctaaataat 481 gcttatgctg ctccacaaac aattgttttt gttcgttatc ttctggacta cccttcaact 541 tttcataatg actagctaaa tataaaaaat tcacatattt gcttggcaga gccagctcat 601 ttcctttttg taattctccg gcactagcca gcatccgttt acgaccgttt tctaactcaa 661 aaagactata tttaggtagt ttaatgatta agtctttttt aacttcctta tatcctttag 721 cttctaaaaa gtcaatcgga tttttttcaa aggaacttct ttccataatt gtgatcccta 781 gtaactcttt aacggatttt aacttcttcg atttcccttt ttccacctta gcaaccacta 841 ggactgaata agctaccgtt ggactatcaa aaccaccata tttttttgga tcccagtctt 901 ttttacgagc aataagcttg tccgaatttc tttttggtaa aattgactcc ttggagaatc 961 cgcctgtctg tacttctgtt ttcttgacaa tattgacttg gggcatggac aatactttgc 1021 gcactgtggc aaaatctcgc cctttatccc agacaatttc tccagtttcc ccattagttt 1081 cgattagagg gcgtttgcga atctctccat ttgcaagtgt aatttctgtt ttgaagaagt 1141 tcatgatatt agagtaaaag aaatattttg cggttgcttt gcctatttct tgctcagact 1201 tagcaatcat tttacgaaca tcataaactt tataatcacc atagacaaac tccgattcaa 1261 gttttggata tttcttaatc aaagcagttc caacgacggc atttagatac gcatcatggg 1321 catgatggta attgttaatc tcacgtactt tatagaattg gaaatctttt cggaagtcag 1381 aaactaattt agattttaag gtaatcactt taacctctcg aataagttta tcattttcat 1441 cgtatttagt attcatgcga ctatccaaaa tttgtgccac atgcttagtg atttggcgag 1501 tttcaaccaa ttggcgtttg ataaaaccag ctttatcaag ttcactcaaa cctccacgtt 1561 cagctttcgt taaattatca aacttacgtt gagtgattaa cttggcgttt agaagttgtc 1621 tccaatagtt tttcatcttt ttgactactt cttcacttgg aacgttatcc gatttaccac 1681 gatttttatc agaacgcgtt aagaccttat tgtctattga atcgtcttta aggaaacttt 1741 gtggaacaat ggcatcgaca tcataatcac ttaaacgatt aatatctaat tcttggtcca 1801 catacatgtc tcttccattt tggagataat agagatagag cttttcattt tgcaattgag 1861 tattttcaac aggatgctct ttaagaatct gacttcctaa ttctttgata ccttcttcga 1921 ttcgtttcat acgctctcgc gaatttttct ggcccttttg agttgtctga ttttcacgtg 1981 ccatttcaat aacgatattt tctggcttat gccgccccat tactttgacc aattcatcaa 2041 caacttttac agtctgtaaa ataccttttt taatagcagg gctaccagct aaatttgcaa 2101 tatgttcatg taaactatcg ccttgtccag acacttgtgc tttttgaatg tcttctttaa 2161 atgtcaaact atcatcatgg atcagctgca taaaattgcg attggcaaaa ccatctgatt 2221 tcaaaaaatc taatattgtt ttgccagatt gcttatccct aataccatta atcaattttc 2281 gagacaaacg tccccaacca gtataacggc gacgtttaag ctgtttcatc accttatcat 2341 caaagaggtg agcatatgtt ttaagtcttt cctcaatcat ctccctatct tcaaataagg 2401 tcaatgttaa aacaatatcc tctaagatat cttcattttc ttcattatcc aaaaaatctt 2461 tatctttaat aatttttagc aaatcatggt aggtacctaa tgaagcatta aatctatctt 2521 caactcctga aatttcaaca ctatcaaaac attctatttt tttgaaataa tcttctttta 2581 attgcttaac ggttactttt cgatttgttt tgaagagtaa atcaacaatg gctttcttct 2641 gttcacctga aagaaatgct ggttttcgca ttccttcagt aacatatttg acctttgtca 2701 attcgttata aaccgtaaaa tactcataaa gcaaactatg ttttggtagt actttttcat 2761 ttggaagatt tttatcaaag tttgtcatgc gttcaataaa tgattgagct gaagcacctt 2821 tatcgacaac ttcttcaaaa ttccatgggg taattgtttc ttcagacttc cgagtcatcc 2881 atgcaaaacg actattgcca cgcgccaatg gaccaacata ataaggaata cgaaaagtca 2941 agattttttc aatcttctca cgattgtctt ttaaaaatgg ataaaagtct tcttgtcttc 3001 tcaaaatagc atgcagctca cccaagtgaa tttgatgggg aatagagccg ttgtcaaagg 3061 tccgttgctt gcgcagcaaa tcttcacgat ttagtttcac caataattcc tcagtaccat 3121 ccattttttc taaaattggt ttgataaatt tataaaattc ttcttggcta gctcccccat 3181 caatataacc tgcatatccg ttttttgatt gatcaaaaaa gatttcttta tacttttctg 3241 gaagttgttg tcgaactaaa gcttttaaaa gagtcaagtc ttgatgatgt tcatcgtagc 3301 gtttaatcat tgaagctgat aggggagcct tagttatttc agtatttact cttaggatat 3361 ctgaaagtaa aatagcatct gataaattct tagctgccaa aaacaaatca gcatattgat 3421 ctccaatttg cgccaataaa ttatctaaat catcatcgta agtatctttt gaaagctgta 3481 atttagcatc ttctgccaaa tcaaaatttg atttaaaatt aggggtcaaa cccaatgaca 3541 aagcaatgag attcccaaat aagccatttt tcttctcacc ggggagctga gcaatgagat 3601 tttctaatcg tcttgattta ctcaatcgtg cagaaagaat cgctttagca tctactccac 3661 ttgcgttaat agggttttct tcaaataatt gattgtaggt ttgtaccaac tggataaata 3721 gtttgtccac atcactatta tcaggattta aatctccctc aatcaaaaaa tgaccacgaa 3781 acttaatcat atgcgctaag gccaaataga ttaagcgcaa atccgcttta tcagtagaat 3841 ctaccaattt ttttcgcaga tgatagatag ttggatattt ctcatgataa gcaacttcat 3901 ctactatatt tccaaaaata ggatgacgtt catgcttctt gtcttcttcc accaaaaaag 3961 actcttcaag tcgatgaaag aaactatcat ctactttcgc catctcattt gaaaaaatct 4021 cctgtagata acaaatacga ttcttccgac gtgtatacct tctacgagct gtccgtttga 4081 ggcgagtcgc ttccgctgtc tctccactgt caaataaaag agcccctata agattttttt 4141 tgatactgtg gcggtctgta tttcccagaa ccttgaactt tttagacgga accttatatt 4201 catcagtgat caccgcccat ccgacgctat ttgtgccgat agctaagcct attgagtatt 4261 tcttatccat agatcctttc tcctctttag atcttttcaa ttcttttctc tatcactgat 4321 agggagtggt aaaataactc tatcaacgat agagtgtcaa caaaaattag gaattaatga 4381 tgtcaagatt agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg 4441 gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat 4501 tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag 4561 ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta 4621 ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt 4681 taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat 4741 gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta 4801 ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac 4861 ctactactga tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag 4921 gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac 4981 ttaaatgtga aagtgggtct taatactaga gccaattatt gaaggccgct aacgcggcct 5041 ttttttgttt ctggtctccc tactagagtt tacggctagc tcagtcctag gtatagtgct 5101 agctactaga gtcacacagg aaagtactag atggcagaat acaaagataa tttacttggt 5161 gaggcgaaca gctttctcga agtgctggaa caggtttcgc atctcgcacc gctggacaaa 5221 ccggtgctca tcatcggcga acgcggcacc ggtaaagagc tgattgccag ccgcctgcat 5281 tatctctcct cccgttggca agggccgttt atttccctta actgcgcggc gttaaatgaa 5341 aatctgctgg attccgaact gtttggtcac gaagcggggg cgtttaccgg tgcgcaaaaa 5401 cgtcatccag ggagatttga acgtgccgac ggcggtacgc tatttcttga tgaactcgct 5461 acggcaccga tgatggtgca ggagaaatta ttgcgcgtga ttgagtacgg tgaactggag 5521 cgcgttggcg gcagccaacc attgcaggtg aatgtgcggt tggtatgcgc gacgaatgcc 5581 gatctcccgg cgatggtcaa tgaaggcact tttcgcgctg acctgctcga ccgactggct 5641 tttgatgttg tacaactgcc accactgcgc gagcgcgaaa gcgacataat gttgatggca 5701 gaatactttg ccatccagat gtgtcgggaa atcaagctgc ctctgttccc ggggtttacg 5761 gagcgcgcca gagaaacatt gctgaattat cgttggccgg gaaatattcg tgaattgaaa 5821 aacgtggtgg aacgttcagt gtatcgccac ggcaccagcg attatccgct tgatgacatc 5881 attattgatc cctttaaacg gcgtccgcct gaagacgcta tcgccgtttc agaaaccacc 5941 tcgcttccaa cactgccgct ggatttacgt gagtttcaga tgcagcagga aaaagagttg 6001 ctgcaactca gtttgcaaat gaatgcacgc acacgccgcc gcgaacgtcg cgcagagaaa 6061 caggctcaat ggaaagcagc aaattaatac tagagccaat tattgaaggc cgctaacgcg 6121 gccttttttt gtttctggtc tccctactag aagttatccc gacgcgccgt ccggtctgag 6181 gtctggaaga tcggttagtg taattcgcta actcatcctg gcatgttgct gttgattctt 6241 caatcagatc tttataaatc aaaaagataa aaaattggca cgcaaattgt attaacagtt 6301 cagcaggaca atcctgaacg caaatactag agagctgtca ccggatgtgc tttccggtct 6361 gatgagtccg tgaggacgaa acagcctcta caaataattt tgtttaacta gagattaaag 6421 aggagaaata ccatatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg 6481 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg 6541 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggtac 6601 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg 6661 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac 6721 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag 6781 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagaa gacggcaata 6841 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata 6901 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg 6961 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc 7021 cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg 7081 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac 7141 tgtacaaaag gcctgctgca aacgacgaaa actacgctgc atcagtttaa taatactaga 7201 gccaattatt gaaggcctcc ctaacggggg gccttttttt gtttctggtc tccctactag 7261 tagcggccgc tgcagtccgg caaaaaaggg caaggtgtca ccaccctgcc ctttttcttt 7321 aaaaccgaaa agattacttc gcgttatgca ggcttcctcg ctcactgact cgctgcgctc 7381 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatct cgaggttaca 7441 ttgtcgatct gttcatggtg aacagctttg aatgcaccaa aaactcgtaa aagctctgat 7501 gtatctatct tttttacacc gttttcatct gtgcatatgg acagttttcc ctttgatatg 7561 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca 7621 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 7681 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 7741 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 7801 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 7861 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 7921 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt 7981 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 8041 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat 8101 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 8161 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa 8221 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa 8281 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc 8341 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct 8401 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct 8461 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 8521 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc 8581 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 8641 tcaatgataa ttacatgtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 8701 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg 8761 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 8821 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 8881 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 8941 aggcttaagt agcaccctcg caagctcggg caaatcgctg aatattcctt ttgtctccga 9001 ccatcaggca cctgagtcgc tgtctttttc gtgacattca gttcgctgcg ctcacggctc 9061 tggcagtgaa tgggggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac 9121 tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc 9181 tatgtggtgc tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct 9241 gaccacttcg gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca 9301 gcggtatcat caacaggctt acccgtctta ctgtccctag tgcttggatt ctcaccaata 9361 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt 9421 actggatcta tcaacaggag tccaagcgag ctcgtaaact tggtctgaca gttaccaatg 9481 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 9541 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 9601 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 9661 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 9721 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 9781 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 9841 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 9901 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 9961 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 10021 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 10081 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 10141 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 10201 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 10261 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 10321 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 10381 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 10441 atttcccatg gtgccacctg acgtctaaga aaccattatt atcatgacat t //