LOCUS Exported 17028 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION piggyBac transposon for dox-inducible expression of the OKMS (Oct4, Klf4[10-483], c-Myc, Sox2) polycistronic reprogramming cassette (with mCherry). KRAB-dCas9 is constitutively expressed.. ACCESSION . VERSION . KEYWORDS PB-TAC-OKMS-KRAB-dCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 17028) 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 17028) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..17028 /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 4226..4285 /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 5681..5746 /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(6786..6810) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 6852..7438 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(7019..7036) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 7246..7265 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 7439..8149 /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(7584..7602) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 7852..7871 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(8161..8180) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 8226..8281 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(8280..8299) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" enhancer 8718..9003 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" intron 9283..9510 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 9522..9531 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 9558..9743 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" CDS 9765..13868 /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" CDS 13872..13898 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 13917..13937 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 13944..13964 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(14031..14048) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 14037..14244 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 14655..14689 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter complement(14838..14856) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(14877..14893) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14877..14893) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(14890..14912) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 14901..14917 /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(14925..14955) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14970..14991 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(15108..15125) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(15279..15867) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(15359..15378) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(16038..16898) /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 16661..16680 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(16899..17003) /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 gtccactcat ggctgtcagc gacgctctgc tcccgtcctt 2821 ctccacgttc gcgtccggcc cggcgggaag ggagaagaca ctgcgtccag caggtgcccc 2881 gactaaccgt tggcgtgagg aactctctca catgaagcga cttcccccac ttcccggccg 2941 cccctacgac ctggcggcga cggtggccac agacctggag agtggcggag ctggtgcagc 3001 ttgcagcagt aacaacccgg ccctcctagc ccggagggag accgaggagt tcaacgacct 3061 cctggaccta gactttatcc tttccaactc gctaacccac caggaatcgg tggccgccac 3121 cgtgaccacc tcggcgtcag cttcatcctc gtcttcccca gcgagcagcg gccctgccag 3181 cgcgccctcc acctgcagct tcagctatcc gatccgggcc gggggtgacc cgggcgtggc 3241 tgccagcaac acaggtggag ggctcctcta cagccgagaa tctgcgccac ctcccacggc 3301 ccccttcaac ctggcggaca tcaatgacgt gagcccctcg ggcggcttcg tggctgagct 3361 cctgcggccg gagttggacc cagtatacat tccgccacag cagcctcagc cgccaggtgg 3421 cgggctgatg ggcaagtttg tgctgaaggc gtctctgacc acccctggca gcgagtacag 3481 cagcccttcg gtcatcagtg ttagcaaagg aagcccagac ggcagccacc ccgtggtagt 3541 ggcgccctac agcggtggcc cgccgcgcat gtgccccaag attaagcaag aggcggtccc 3601 gtcctgcacg gtcagccggt ccctagaggc ccatttgagc gctggacccc agctcagcaa 3661 cggccaccgg cccaacacac acgacttccc cctggggcgg cagctcccca ccaggactac 3721 ccctacactg agtcccgagg aactgctgaa cagcagggac tgtcaccctg gcctgcctct 3781 tcccccagga ttccatcccc atccggggcc caactaccct cctttcctgc cagaccagat 3841 gcagtcacaa gtcccctctc tccattatca agagctcatg ccaccgggtt cctgcctgcc 3901 agaggagccc aagccaaaga ggggaagaag gtcgtggccc cggaaaagaa cagccaccca 3961 cacttgtgac tatgcaggct gtggcaaaac ctataccaag agttctcatc tcaaggcaca 4021 cctgcgaact cacacaggcg agaaacctta ccactgtgac tgggacggct gtgggtggaa 4081 attcgcccgc tccgatgaac tgaccaggca ctaccgcaaa cacacagggc accggccctt 4141 tcagtgccag aagtgcgaca gggccttttc caggtcggac caccttgcct tacacatgaa 4201 gaggcacttt ctcgagggct cgggccagtg tactaattat gctctcttga aattggctgg 4261 agatgttgag agcaacccag gtcccggcgc gccactggat ttcctttggg cgttggaaac 4321 cccgcagaca gccacgacga tgcccctcaa cgtgaacttc accaacagga actatgacct 4381 cgactacgac tccgtacagc cctatttcat ctgcgacgag gaagagaatt tctatcacca 4441 gcaacagcag agcgagctgc agccgcccgc gcccagtgag gatatctgga agaaattcga 4501 gctgcttccc accccgcccc tgtccccgag ccgccgctcc gggctctgct ctccatccta 4561 tgttgcggtc gctacgtcct tctccccaag ggaagacgat gacggcggcg gtggcaactt 4621 ctccaccgcc gatcagctgg agatgatgac cgagttactt ggaggagaca tggtgaacca 4681 gagcttcatc tgcgatcctg acgacgagac cttcatcaag aacatcatca tccaggactg 4741 tatgtggagc ggtttctcag ccgctgccaa gctggtctcg gagaagctgg cctcctacca 4801 ggctgcgcgc aaagacagca ccagcctgag ccccgcccgc gggcacagcg tctgctccac 4861 ctccagcctg tacctgcagg acctcaccgc cgccgcgtcc gagtgcattg acccctcagt 4921 ggtctttccc tacccgctca acgacagcag ctcgcccaaa tcctgtacct cgtccgattc 4981 cacggccttc tctccttcct cggactcgct gctgtcctcc gagtcctccc cacgggccag 5041 ccctgagccc ctagtgctgc atgaggagac accgcccacc accagcagcg actctgaaga 5101 agagcaagaa gatgaggaag aaattgatgt ggtgtctgtg gagaagaggc aaacccctgc 5161 caagaggtcg gagtcgggct catctccatc ccgaggccac agcaaacctc cgcacagccc 5221 actggtcctc aagaggtgcc acgtctccac tcaccagcac aactacgccg cacccccctc 5281 cacaaggaag gactatccag ctgccaagag ggccaagttg gacagtggca gggtcctgaa 5341 gcagatcagc aacaaccgca agtgctccag ccccaggtcc tcagacacgg aggaaaacga 5401 caagaggcgg acacacaacg tcttggaacg tcagaggagg aacgagctga agcgcagctt 5461 ttttgccctg cgtgaccaga tccctgaatt ggaaaacaac gaaaaggccc ccaaggtagt 5521 gatcctcaaa aaagccaccg cctacatcct gtccattcaa gcagacgagc acaagctcac 5581 ctctgaaaag gacttattga ggaaacgacg agaacagttg aaacacaaac tcgaacagct 5641 tcgaaactct ggtgcagggg atctcaaaat tgtcgctcct gtcaaacaaa ctcttaactt 5701 tgatttactc aaactggctg gggatgtaga aagcaatcca ggtccactca tgaacaattc 5761 gcccttcacc atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc 5821 ttcggggggc ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa 5881 gaacagcccg gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca 5941 gcggcgtaag atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct 6001 gggcgcggag tggaaacttt tgtccgagac cgagaagcgg ccgttcatcg acgaggccaa 6061 gcggctgcgc gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcggaa 6121 aaccaagacg ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg 6181 cgggaacagc atggcgagcg gggttggggt gggcgccggc ctgggtgcgg gcgtgaacca 6241 gcgcatggac agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca 6301 ggagcagctg ggctacccgc agcacccggg cctcaacgct cacggcgcgg cacagatgca 6361 accgatgcac cgctacgacg tcagcgccct gcagtacaac tccatgacca gctcgcagac 6421 ctacatgaac ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat 6481 ggcgctgggc tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt 6541 tacctcttcc tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag 6601 catgtacctc cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc 6661 ccagcactac cagagcggcc cggtgcccgg cacggccatt aacggcacac tgcccctgtc 6721 gcacatgtga gggctggaag ggcgaattgc ctgcaggaat tcgcggccgc actcgagata 6781 tctagaccca gctttcttgt acaaagtggt gatctagcta aaacgcggcc tcgaatcgaa 6841 ttccgccccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa 6901 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat 6961 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct 7021 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct 7081 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc 7141 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa 7201 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc 7261 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg 7321 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc 7381 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat 7441 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt 7501 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 7561 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt 7621 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc 7681 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt 7741 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 7801 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat 7861 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc 7921 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga 7981 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa 8041 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg 8101 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaaa gcggccgcga 8161 tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc 8221 tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc 8281 actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta 8341 gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg 8401 tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac 8461 ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa 8521 attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat 8581 agctgtccct cttctcttat ggagatccct cgacctgcag gaattcgatt acttaagcct 8641 aggatccaga tctgtacatt cgaatcgatg aattcgttaa ccatggaagc ttctgcagcg 8701 gccgcactag aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 8761 aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa 8821 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 8881 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac 8941 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 9001 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc 9061 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg 9121 ggggggcgcg cgccrggsgg ggsggggsgg ggsggggggs ggggsggggs grggsggrgr 9181 ggkgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg 9241 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc 9301 cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc 9361 gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg 9421 agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg 9481 gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg gatgctaagt 9541 cactaactgc ctggtcccgg acactggtga ccttcaagga tgtatttgtg gacttcacca 9601 gggaggagtg gaagctgctg gacactgctc agcagatcgt gtacagaaat gtgatgctgg 9661 agaactataa gaacctggtt tccttgggtt atcagcttac taagccagat gtgatcctcc 9721 ggttggagaa gggagaagag cccggcggtt ccggcggagg gtcgatggac aagaagtaca 9781 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca 9841 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga 9901 acctgatcgg cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga 9961 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct 10021 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc 10081 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg 10141 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca 10201 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg 10261 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca 10321 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg 10381 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga 10441 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc 10501 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc 10561 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt 10621 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc 10681 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg 10741 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga 10801 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg 10861 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca 10921 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg 10981 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc 11041 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct 11101 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga 11161 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg 11221 ccagcgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga 11281 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca 11341 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa 11401 aagccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag 11461 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc 11521 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact 11581 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt 11641 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca 11701 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc 11761 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg 11821 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag 11881 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca 11941 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg 12001 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag 12061 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag 12121 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc 12181 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc 12241 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgctatcgtg cctcagagct 12301 ttctgaagga cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca 12361 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc 12421 tgctgaatgc caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag 12481 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc 12541 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga 12601 acgacaaact gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt 12661 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg 12721 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa 12781 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg 12841 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt 12901 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa 12961 acggcgaaac aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag 13021 tgctgtctat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca 13081 gcaaagagtc tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact 13141 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg 13201 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga 13261 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg 13321 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc 13381 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac 13441 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga 13501 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc 13561 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta 13621 atctggacaa ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg 13681 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt 13741 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca 13801 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg 13861 gaggcgacgc ctatccctat gacgtgcccg attatgccag cctgggcagc ggctccccca 13921 agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa agtggacggc attggtagtg 13981 ggagcaacgg cagcagcgga tcctaagaat tcctagagct cgctgatcag cctcgactgt 14041 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 14101 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 14161 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 14221 agagaatagc aggcatgctg gggagcggcc gcactagtct agtctagagc gatcgcccgg 14281 gtttaaacgc tagccaacaa gctcgtcatc gctttgcaga agagcagaga ggatatgctc 14341 atcgtctaaa gaactaccca ttttattata tattagtcac gatatctata acaagaaaat 14401 atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga 14461 gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt 14521 atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc 14581 ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca 14641 atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taaaaaagat 14701 ttgcgcttta ctcgacctaa actttaaaca cgtcatagaa tcttcgtttg acaaaaacca 14761 cattgtggcc aagctgtgtg acgcgacgcg cgctaaagaa tggcaaacca agtcgcgcga 14821 ggtaccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca 14881 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 14941 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 15001 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 15061 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 15121 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 15181 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 15241 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 15301 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 15361 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 15421 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 15481 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 15541 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 15601 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 15661 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 15721 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 15781 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 15841 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 15901 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 15961 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 16021 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 16081 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 16141 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 16201 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 16261 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 16321 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 16381 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 16441 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 16501 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 16561 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 16621 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 16681 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 16741 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 16801 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 16861 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 16921 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 16981 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacct //