LOCUS Exported 16845 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION piggyBac transposon for dox-inducible expression of the OK+9MS (Oct4, Klf4[1-483], c-Myc, Sox2) polycistronic reprogramming cassette (with mCherry). SpCas9 is constitutively expressed.. ACCESSION . VERSION . KEYWORDS PB-TAC-OK+9MS-Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16845) AUTHORS Kagawa H, Shimamoto R, Kim SI, Oceguera-Yanez F, Yamamoto T, Schroeder T, Woltjen K TITLE OVOL1 Influences the Determination and Expansion of iPSC Reprogramming Intermediates. JOURNAL Stem Cell Reports. 2018 Dec 28. pii: S2213-6711(18)30527-7. doi: 10.1016/j.stemcr.2018.12.008. PUBMED 30639212 REFERENCE 2 (bases 1 to 16845) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16845 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(1..456) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(138..159) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 350..369 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 583..605 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 597..614 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 598..614 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 621..640 /label=T7 /note="T7 promoter, forward primer" promoter 621..639 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 706..768 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" protein_bind 1140..1158 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1177..1195 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1214..1232 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1251..1269 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1288..1306 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1325..1343 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1362..1380 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1399..1417 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 1439..1463 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 1543..1567 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2720..2785 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" CDS 4250..4309 /codon_start=1 /product="2A peptide from equine rhinitis A virus polyprotein" /label=E2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="QCTNYALLKLAGDVESNPGP" CDS 5705..5770 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" protein_bind complement(6810..6834) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 6876..7462 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(7043..7060) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 7270..7289 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 7463..8173 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(7608..7626) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 7876..7895 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(8185..8204) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 8250..8305 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(8304..8323) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" enhancer 8742..9027 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 9029..9306 /label=chicken beta-actin promoter intron 9307..9534 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 9546..9555 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 9555..9620 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 9627..9647 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9672..13772 /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 13773..13820 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(13848..13865) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 13854..14061 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 14472..14506 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter complement(14655..14673) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(14694..14710) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14694..14710) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(14707..14729) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 14718..14734 /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)." promoter complement(14742..14772) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14787..14808 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(14925..14942) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(15096..15684) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(15176..15195) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(15855..16715) /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 16478..16497 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(16716..16820) /gene="bla" /label=AmpR promoter ORIGIN 1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca 481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag ctcggtattc 661 acgacagcag gctgaataat aaaaaaatta gaaactatta tttaacccta gaaagataat 721 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt 781 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact 841 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa 901 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac 961 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta 1021 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg 1081 cacatatcaa tattatgctc tcgttaatta actcgaggga tccaagctgg ccgcacgtct 1141 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta 1201 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag 1261 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc 1321 gagctcccta tcagtgatag agaaggtacg tctagaacgt ctccctatca gtgatagaga 1381 agtcgacacg tctcgagctc cctatcagtg atagagaagg taccccctat ataagcagag 1441 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata 1501 gaagacaccg ggaccgatcc agcctccgcg gtggcggcca tcacaagttt gtacaaaaaa 1561 gcaggctggc gccggaacca attcagtcga ctggatccgg taccgaattc gcccttaaaa 1621 agcaggctcc accttcccca tggctggaca cctggcttca gacttcgcct tctcaccccc 1681 accaggtggg ggtgatgggt cagcagggct ggagccgggc tgggtggatt ctcgaacctg 1741 gctaagcttc caagggcctc caggtgggcc tggaatcgga ccaggctcag aggtattggg 1801 gatctcccca tgtccgcccg catacgagtt ctgcggaggg atggcatact gtggacctca 1861 ggttggactg ggcctagtcc cccaagttgg cgtggagact ttgcagcctg agggccaggc 1921 aggagcacga gtggaaagca actcagaggg aacctcctct gagccctgtg ccgaccgccc 1981 caatgccgtg aagttggaga aggtggaacc aactcccgag gagtcccagg acatgaaagc 2041 cctgcagaag gagctagaac agtttgccaa gctgctgaag cagaagagga tcaccttggg 2101 gtacacccag gccgacgtgg ggctcaccct gggcgttctc tttggaaagg tgttcagcca 2161 gaccaccatc tgtcgcttcg aggccttgca gctcagcctt aagaacatgt gtaagctgcg 2221 gcccctgctg gagaagtggg tggaggaagc cgacaacaat gagaaccttc aggagatatg 2281 caaatcggag accctggtgc aggcccggaa gagaaagcga actagcattg agaaccgtgt 2341 gaggtggagt ctggagacca tgtttctgaa gtgcccgaag ccctccctac agcagatcac 2401 tcacatcgcc aatcagcttg ggctagagaa ggatgtggtt cgagtatggt tctgtaaccg 2461 gcgccagaag ggcaaaagat caagtattga gtattcccaa cgagaagagt atgaggctac 2521 agggacacct ttcccagggg gggctgtatc ctttcctctg cccccaggtc cccactttgg 2581 caccccaggc tatggaagcc cccacttcac cacactctac tcagtccctt ttcctgaggg 2641 cgaggccttt ccctctgttc ccgtcactgc tctgggctct cccatgcatt caaacgggga 2701 tctcaaaatt gtcgctcctg tcaaacaaac tcttaacttt gatttactca aactggctgg 2761 ggatgtagaa agcaatccag gtccaatgag gcagccacct ggcgagtctg acatggctgt 2821 cagcgacgct ctgctcccgt ccttctccac gttcgcgtcc ggcccggcgg gaagggagaa 2881 gacactgcgt ccagcaggtg ccccgactaa ccgttggcgt gaggaactct ctcacatgaa 2941 gcgacttccc ccacttcccg gccgccccta cgacctggcg gcgacggtgg ccacagacct 3001 ggagagtggc ggagctggtg cagcttgcag cagtaacaac ccggccctcc tagcccggag 3061 ggagaccgag gagttcaacg acctcctgga cctagacttt atcctttcca actcgctaac 3121 ccaccaggaa tcggtggccg ccaccgtgac cacctcggcg tcagcttcat cctcgtcttc 3181 cccagcgagc agcggccctg ccagcgcgcc ctccacctgc agcttcagct atccgatccg 3241 ggccgggggt gacccgggcg tggctgccag caacacaggt ggagggctcc tctacagccg 3301 agaatctgcg ccacctccca cggccccctt caacctggcg gacatcaatg acgtgagccc 3361 ctcgggcggc ttcgtggctg agctcctgcg gccggagttg gacccagtat acattccgcc 3421 acagcagcct cagccgccag gtggcgggct gatgggcaag tttgtgctga aggcgtctct 3481 gaccacccct ggcagcgagt acagcagccc ttcggtcatc agtgttagca aaggaagccc 3541 agacggcagc caccccgtgg tagtggcgcc ctacagcggt ggcccgccgc gcatgtgccc 3601 caagattaag caagaggcgg tcccgtcctg cacggtcagc cggtccctag aggcccattt 3661 gagcgctgga ccccagctca gcaacggcca ccggcccaac acacacgact tccccctggg 3721 gcggcagctc cccaccagga ctacccctac actgagtccc gaggaactgc tgaacagcag 3781 ggactgtcac cctggcctgc ctcttccccc aggattccat ccccatccgg ggcccaacta 3841 ccctcctttc ctgccagacc agatgcagtc acaagtcccc tctctccatt atcaagagct 3901 catgccaccg ggttcctgcc tgccagagga gcccaagcca aagaggggaa gaaggtcgtg 3961 gccccggaaa agaacagcca cccacacttg tgactatgca ggctgtggca aaacctatac 4021 caagagttct catctcaagg cacacctgcg aactcacaca ggcgagaaac cttaccactg 4081 tgactgggac ggctgtgggt ggaaattcgc ccgctccgat gaactgacca ggcactaccg 4141 caaacacaca gggcaccggc cctttcagtg ccagaagtgc gacagggcct tttccaggtc 4201 ggaccacctt gccttacaca tgaagaggca ctttctcgag ggctcgggcc agtgtactaa 4261 ttatgctctc ttgaaattgg ctggagatgt tgagagcaac ccaggtcccg gcgcgccact 4321 ggatttcctt tgggcgttgg aaaccccgca gacagccacg acgatgcccc tcaacgtgaa 4381 cttcaccaac aggaactatg acctcgacta cgactccgta cagccctatt tcatctgcga 4441 cgaggaagag aatttctatc accagcaaca gcagagcgag ctgcagccgc ccgcgcccag 4501 tgaggatatc tggaagaaat tcgagctgct tcccaccccg cccctgtccc cgagccgccg 4561 ctccgggctc tgctctccat cctatgttgc ggtcgctacg tccttctccc caagggaaga 4621 cgatgacggc ggcggtggca acttctccac cgccgatcag ctggagatga tgaccgagtt 4681 acttggagga gacatggtga accagagctt catctgcgat cctgacgacg agaccttcat 4741 caagaacatc atcatccagg actgtatgtg gagcggtttc tcagccgctg ccaagctggt 4801 ctcggagaag ctggcctcct accaggctgc gcgcaaagac agcaccagcc tgagccccgc 4861 ccgcgggcac agcgtctgct ccacctccag cctgtacctg caggacctca ccgccgccgc 4921 gtccgagtgc attgacccct cagtggtctt tccctacccg ctcaacgaca gcagctcgcc 4981 caaatcctgt acctcgtccg attccacggc cttctctcct tcctcggact cgctgctgtc 5041 ctccgagtcc tccccacggg ccagccctga gcccctagtg ctgcatgagg agacaccgcc 5101 caccaccagc agcgactctg aagaagagca agaagatgag gaagaaattg atgtggtgtc 5161 tgtggagaag aggcaaaccc ctgccaagag gtcggagtcg ggctcatctc catcccgagg 5221 ccacagcaaa cctccgcaca gcccactggt cctcaagagg tgccacgtct ccactcacca 5281 gcacaactac gccgcacccc cctccacaag gaaggactat ccagctgcca agagggccaa 5341 gttggacagt ggcagggtcc tgaagcagat cagcaacaac cgcaagtgct ccagccccag 5401 gtcctcagac acggaggaaa acgacaagag gcggacacac aacgtcttgg aacgtcagag 5461 gaggaacgag ctgaagcgca gcttttttgc cctgcgtgac cagatccctg aattggaaaa 5521 caacgaaaag gcccccaagg tagtgatcct caaaaaagcc accgcctaca tcctgtccat 5581 tcaagcagac gagcacaagc tcacctctga aaaggactta ttgaggaaac gacgagaaca 5641 gttgaaacac aaactcgaac agcttcgaaa ctctggtgca ggggatctca aaattgtcgc 5701 tcctgtcaaa caaactctta actttgattt actcaaactg gctggggatg tagaaagcaa 5761 tccaggtcca ctcatgaaca attcgccctt caccatgtat aacatgatgg agacggagct 5821 gaagccgccg ggcccgcagc aagcttcggg gggcggcggc ggaggaggca acgccacggc 5881 ggcggcgacc ggcggcaacc agaagaacag cccggaccgc gtcaagaggc ccatgaacgc 5941 cttcatggta tggtcccggg ggcagcggcg taagatggcc caggagaacc ccaagatgca 6001 caactcggag atcagcaagc gcctgggcgc ggagtggaaa cttttgtccg agaccgagaa 6061 gcggccgttc atcgacgagg ccaagcggct gcgcgctctg cacatgaagg agcacccgga 6121 ttataaatac cggccgcggc ggaaaaccaa gacgctcatg aagaaggata agtacacgct 6181 tcccggaggc ttgctggccc ccggcgggaa cagcatggcg agcggggttg gggtgggcgc 6241 cggcctgggt gcgggcgtga accagcgcat ggacagctac gcgcacatga acggctggag 6301 caacggcagc tacagcatga tgcaggagca gctgggctac ccgcagcacc cgggcctcaa 6361 cgctcacggc gcggcacaga tgcaaccgat gcaccgctac gacgtcagcg ccctgcagta 6421 caactccatg accagctcgc agacctacat gaacggctcg cccacctaca gcatgtccta 6481 ctcgcagcag ggcacccccg gtatggcgct gggctccatg ggctctgtgg tcaagtccga 6541 ggccagctcc agcccccccg tggttacctc ttcctcccac tccagggcgc cctgccaggc 6601 cggggacctc cgggacatga tcagcatgta cctccccggc gccgaggtgc cggagcccgc 6661 tgcgcccagt agactgcaca tggcccagca ctaccagagc ggcccggtgc ccggcacggc 6721 cattaacggc acactgcccc tgtcgcacat gtgagggctg gaagggcgaa ttgcctgcag 6781 gaattcgcgg ccgcactcga gatatctaga cccagctttc ttgtacaaag tggtgatcta 6841 gctaaaacgc ggcctcgaat cgaattccgc ccccccccct ctccctcccc cccccctaac 6901 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 6961 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 7021 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 7081 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 7141 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 7201 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 7261 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 7321 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 7381 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 7441 cgatgataat atggccacaa ccatggtgag caagggcgag gaggataaca tggccatcat 7501 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga 7561 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt 7621 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg 7681 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc 7741 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac 7801 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa 7861 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga 7921 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa 7981 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca 8041 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta 8101 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga 8161 gctgtacaag taaagcggcc gcgatctttt tccctctgcc aaaaattatg gggacatcat 8221 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt 8281 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa 8341 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat 8401 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc 8461 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt 8521 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct 8581 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct 8641 gcaggaattc gattacttaa gcctaggatc cagatctgta cattcgaatc gatgaattcg 8701 ttaaccatgg aagcttctgc agcggccgca ctagaggtac ccgttacata acttacggta 8761 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca 8821 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 8881 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 8941 cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 9001 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc 9061 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt 9121 gcagcgatgg gggcgggggg gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg 9181 gggsggggsg gggsgrggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 9241 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 9301 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc 9361 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct 9421 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt 9481 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga 9541 ccggtgccac catggactat aaggaccacg acggagacta caaggatcat gatattgatt 9601 acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag 9661 tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct 9721 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca 9781 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa 9841 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc 9901 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct 9961 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca 10021 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 10081 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc 10141 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca 10201 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 10261 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca 10321 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt 10381 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc 10441 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc 10501 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg 10561 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga 10621 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc 10681 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg 10741 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc 10801 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc 10861 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag 10921 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg 10981 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg 11041 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact 11101 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 11161 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 11221 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 11281 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 11341 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 11401 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 11461 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 11521 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 11581 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 11641 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga 11701 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc 11761 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg 11821 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc 11881 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga 11941 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 12001 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 12061 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 12121 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 12181 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 12241 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 12301 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 12361 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 12421 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc 12481 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc 12541 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga 12601 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga 12661 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg 12721 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct 12781 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 12841 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 12901 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 12961 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 13021 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 13081 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 13141 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 13201 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 13261 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 13321 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 13381 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 13441 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 13501 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 13561 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 13621 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 13681 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 13741 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg 13801 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc 13861 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 13921 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 13981 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 14041 gaatagcagg catgctgggg agcggccgca ctagtctagt ctagagcgat cgcccgggtt 14101 taaacgctag ccaacaagct cgtcatcgct ttgcagaaga gcagagagga tatgctcatc 14161 gtctaaagaa ctacccattt tattatatat tagtcacgat atctataaca agaaaatata 14221 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt 14281 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata 14341 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc 14401 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata 14461 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa aaaagatttg 14521 cgctttactc gacctaaact ttaaacacgt catagaatct tcgtttgaca aaaaccacat 14581 tgtggccaag ctgtgtgacg cgacgcgcgc taaagaatgg caaaccaagt cgcgcgaggt 14641 accagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag 14701 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 14761 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 14821 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 14881 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 14941 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 15001 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 15061 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 15121 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 15181 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 15241 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 15301 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 15361 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 15421 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 15481 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 15541 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 15601 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 15661 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 15721 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 15781 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 15841 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 15901 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 15961 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 16021 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 16081 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 16141 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 16201 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 16261 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 16321 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 16381 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 16441 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 16501 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 16561 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 16621 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 16681 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 16741 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 16801 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacct //