LOCUS Exported 16826 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). SpCas9 is constitutively expressed.. ACCESSION . VERSION . KEYWORDS PB-TAC-OKMS-Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16826) 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 16826) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16826 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 588..610 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 602..619 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 603..619 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 711..773 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" protein_bind 1145..1163 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1182..1200 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1219..1237 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1256..1274 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1293..1311 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1330..1348 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1367..1385 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1404..1422 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 1444..1468 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 1548..1572 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2725..2790 /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 4231..4290 /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 5686..5751 /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(6791..6815) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 6857..7442 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(7024..7041) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 7251..7270 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 7443..8153 /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(7588..7606) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 7856..7875 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(8165..8184) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 8230..8285 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(8284..8303) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" enhancer 8720..9005 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 9007..9284 /label=chicken beta-actin promoter intron 9285..9513 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 9525..9534 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 9534..9599 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 9606..9626 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9651..13751 /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 13752..13799 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(13827..13844) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 13833..14040 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 14451..14485 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter complement(14635..14653) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(14674..14690) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14674..14690) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(14687..14709) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 14698..14714 /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(14722..14752) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14767..14788 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(14905..14922) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(15076..15664) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(15156..15175) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(15835..16695) /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 16458..16477 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(16696..16800) /gene="bla" /label=AmpR promoter ORIGIN 1 ggaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cgccattcgc cattcaggct 481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgg 661 tattcacgac agcaggctga ataataaaaa aattagaaac tattatttaa ccctagaaag 721 ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta 781 tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt 841 acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa 901 aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa cattctctct 961 tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta taaaataagt 1021 aattagctta acctatacat aatagaaaca aattatactt attagtcagt cagaaacaac 1081 tttggcacat atcaatatta tgctctcgtt aattaactcg agggatccaa gctggccgca 1141 cgtctcccta tcagtgatag agaagtcgac acgtctcgag ctccctatca gtgatagaga 1201 aggtacgtct agaacgtctc cctatcagtg atagagaagt cgacacgtct cgagctccct 1261 atcagtgata gagaaggtac gtctagaacg tctccctatc agtgatagag aagtcgacac 1321 gtctcgagct ccctatcagt gatagagaag gtacgtctag aacgtctccc tatcagtgat 1381 agagaagtcg acacgtctcg agctccctat cagtgataga gaaggtaccc cctatataag 1441 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct 1501 ccatagaaga caccgggacc gatccagcct ccgcggtggc ggccatcaca agtttgtaca 1561 aaaaagcagg ctggcgccgg aaccaattca gtcgactgga tccggtaccg aattcgccct 1621 taaaaagcag gctccacctt ccccatggct ggacacctgg cttcagactt cgccttctca 1681 cccccaccag gtgggggtga tgggtcagca gggctggagc cgggctgggt ggattctcga 1741 acctggctaa gcttccaagg gcctccaggt gggcctggaa tcggaccagg ctcagaggta 1801 ttggggatct ccccatgtcc gcccgcatac gagttctgcg gagggatggc atactgtgga 1861 cctcaggttg gactgggcct agtcccccaa gttggcgtgg agactttgca gcctgagggc 1921 caggcaggag cacgagtgga aagcaactca gagggaacct cctctgagcc ctgtgccgac 1981 cgccccaatg ccgtgaagtt ggagaaggtg gaaccaactc ccgaggagtc ccaggacatg 2041 aaagccctgc agaaggagct agaacagttt gccaagctgc tgaagcagaa gaggatcacc 2101 ttggggtaca cccaggccga cgtggggctc accctgggcg ttctctttgg aaaggtgttc 2161 agccagacca ccatctgtcg cttcgaggcc ttgcagctca gccttaagaa catgtgtaag 2221 ctgcggcccc tgctggagaa gtgggtggag gaagccgaca acaatgagaa ccttcaggag 2281 atatgcaaat cggagaccct ggtgcaggcc cggaagagaa agcgaactag cattgagaac 2341 cgtgtgaggt ggagtctgga gaccatgttt ctgaagtgcc cgaagccctc cctacagcag 2401 atcactcaca tcgccaatca gcttgggcta gagaaggatg tggttcgagt atggttctgt 2461 aaccggcgcc agaagggcaa aagatcaagt attgagtatt cccaacgaga agagtatgag 2521 gctacaggga cacctttccc agggggggct gtatcctttc ctctgccccc aggtccccac 2581 tttggcaccc caggctatgg aagcccccac ttcaccacac tctactcagt cccttttcct 2641 gagggcgagg cctttccctc tgttcccgtc actgctctgg gctctcccat gcattcaaac 2701 ggggatctca aaattgtcgc tcctgtcaaa caaactctta actttgattt actcaaactg 2761 gctggggatg tagaaagcaa tccaggtcca ctcatggctg tcagcgacgc tctgctcccg 2821 tccttctcca cgttcgcgtc cggcccggcg ggaagggaga agacactgcg tccagcaggt 2881 gccccgacta accgttggcg tgaggaactc tctcacatga agcgacttcc cccacttccc 2941 ggccgcccct acgacctggc ggcgacggtg gccacagacc tggagagtgg cggagctggt 3001 gcagcttgca gcagtaacaa cccggccctc ctagcccgga gggagaccga ggagttcaac 3061 gacctcctgg acctagactt tatcctttcc aactcgctaa cccaccagga atcggtggcc 3121 gccaccgtga ccacctcggc gtcagcttca tcctcgtctt ccccagcgag cagcggccct 3181 gccagcgcgc cctccacctg cagcttcagc tatccgatcc gggccggggg tgacccgggc 3241 gtggctgcca gcaacacagg tggagggctc ctctacagcc gagaatctgc gccacctccc 3301 acggccccct tcaacctggc ggacatcaat gacgtgagcc cctcgggcgg cttcgtggct 3361 gagctcctgc ggccggagtt ggacccagta tacattccgc cacagcagcc tcagccgcca 3421 ggtggcgggc tgatgggcaa gtttgtgctg aaggcgtctc tgaccacccc tggcagcgag 3481 tacagcagcc cttcggtcat cagtgttagc aaaggaagcc cagacggcag ccaccccgtg 3541 gtagtggcgc cctacagcgg tggcccgccg cgcatgtgcc ccaagattaa gcaagaggcg 3601 gtcccgtcct gcacggtcag ccggtcccta gaggcccatt tgagcgctgg accccagctc 3661 agcaacggcc accggcccaa cacacacgac ttccccctgg ggcggcagct ccccaccagg 3721 actaccccta cactgagtcc cgaggaactg ctgaacagca gggactgtca ccctggcctg 3781 cctcttcccc caggattcca tccccatccg gggcccaact accctccttt cctgccagac 3841 cagatgcagt cacaagtccc ctctctccat tatcaagagc tcatgccacc gggttcctgc 3901 ctgccagagg agcccaagcc aaagagggga agaaggtcgt ggccccggaa aagaacagcc 3961 acccacactt gtgactatgc aggctgtggc aaaacctata ccaagagttc tcatctcaag 4021 gcacacctgc gaactcacac aggcgagaaa ccttaccact gtgactggga cggctgtggg 4081 tggaaattcg cccgctccga tgaactgacc aggcactacc gcaaacacac agggcaccgg 4141 ccctttcagt gccagaagtg cgacagggcc ttttccaggt cggaccacct tgccttacac 4201 atgaagaggc actttctcga gggctcgggc cagtgtacta attatgctct cttgaaattg 4261 gctggagatg ttgagagcaa cccaggtccc ggcgcgccac tggatttcct ttgggcgttg 4321 gaaaccccgc agacagccac gacgatgccc ctcaacgtga acttcaccaa caggaactat 4381 gacctcgact acgactccgt acagccctat ttcatctgcg acgaggaaga gaatttctat 4441 caccagcaac agcagagcga gctgcagccg cccgcgccca gtgaggatat ctggaagaaa 4501 ttcgagctgc ttcccacccc gcccctgtcc ccgagccgcc gctccgggct ctgctctcca 4561 tcctatgttg cggtcgctac gtccttctcc ccaagggaag acgatgacgg cggcggtggc 4621 aacttctcca ccgccgatca gctggagatg atgaccgagt tacttggagg agacatggtg 4681 aaccagagct tcatctgcga tcctgacgac gagaccttca tcaagaacat catcatccag 4741 gactgtatgt ggagcggttt ctcagccgct gccaagctgg tctcggagaa gctggcctcc 4801 taccaggctg cgcgcaaaga cagcaccagc ctgagccccg cccgcgggca cagcgtctgc 4861 tccacctcca gcctgtacct gcaggacctc accgccgccg cgtccgagtg cattgacccc 4921 tcagtggtct ttccctaccc gctcaacgac agcagctcgc ccaaatcctg tacctcgtcc 4981 gattccacgg ccttctctcc ttcctcggac tcgctgctgt cctccgagtc ctccccacgg 5041 gccagccctg agcccctagt gctgcatgag gagacaccgc ccaccaccag cagcgactct 5101 gaagaagagc aagaagatga ggaagaaatt gatgtggtgt ctgtggagaa gaggcaaacc 5161 cctgccaaga ggtcggagtc gggctcatct ccatcccgag gccacagcaa acctccgcac 5221 agcccactgg tcctcaagag gtgccacgtc tccactcacc agcacaacta cgccgcaccc 5281 ccctccacaa ggaaggacta tccagctgcc aagagggcca agttggacag tggcagggtc 5341 ctgaagcaga tcagcaacaa ccgcaagtgc tccagcccca ggtcctcaga cacggaggaa 5401 aacgacaaga ggcggacaca caacgtcttg gaacgtcaga ggaggaacga gctgaagcgc 5461 agcttttttg ccctgcgtga ccagatccct gaattggaaa acaacgaaaa ggcccccaag 5521 gtagtgatcc tcaaaaaagc caccgcctac atcctgtcca ttcaagcaga cgagcacaag 5581 ctcacctctg aaaaggactt attgaggaaa cgacgagaac agttgaaaca caaactcgaa 5641 cagcttcgaa actctggtgc aggggatctc aaaattgtcg ctcctgtcaa acaaactctt 5701 aactttgatt tactcaaact ggctggggat gtagaaagca atccaggtcc actcatgaac 5761 aattcgccct tcaccatgta taacatgatg gagacggagc tgaagccgcc gggcccgcag 5821 caagcttcgg ggggcggcgg cggaggaggc aacgccacgg cggcggcgac cggcggcaac 5881 cagaagaaca gcccggaccg cgtcaagagg cccatgaacg ccttcatggt atggtcccgg 5941 gggcagcggc gtaagatggc ccaggagaac cccaagatgc acaactcgga gatcagcaag 6001 cgcctgggcg cggagtggaa acttttgtcc gagaccgaga agcggccgtt catcgacgag 6061 gccaagcggc tgcgcgctct gcacatgaag gagcacccgg attataaata ccggccgcgg 6121 cggaaaacca agacgctcat gaagaaggat aagtacacgc ttcccggagg cttgctggcc 6181 cccggcggga acagcatggc gagcggggtt ggggtgggcg ccggcctggg tgcgggcgtg 6241 aaccagcgca tggacagcta cgcgcacatg aacggctgga gcaacggcag ctacagcatg 6301 atgcaggagc agctgggcta cccgcagcac ccgggcctca acgctcacgg cgcggcacag 6361 atgcaaccga tgcaccgcta cgacgtcagc gccctgcagt acaactccat gaccagctcg 6421 cagacctaca tgaacggctc gcccacctac agcatgtcct actcgcagca gggcaccccc 6481 ggtatggcgc tgggctccat gggctctgtg gtcaagtccg aggccagctc cagccccccc 6541 gtggttacct cttcctccca ctccagggcg ccctgccagg ccggggacct ccgggacatg 6601 atcagcatgt acctccccgg cgccgaggtg ccggagcccg ctgcgcccag tagactgcac 6661 atggcccagc actaccagag cggcccggtg cccggcacgg ccattaacgg cacactgccc 6721 ctgtcgcaca tgtgagggct ggaagggcga attgcctgca ggaattcgcg gccgcactcg 6781 agatatctag acccagcttt cttgtacaaa gtggtgatct agctaaaacg cggcctcgaa 6841 tcgaattccg cccccccccc tctccctccc ccccccctaa cgttactggc cgaagccgct 6901 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg 6961 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt 7021 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg 7081 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac 7141 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg 7201 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct 7261 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg 7321 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aacgtctagg 7381 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa 7441 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 7501 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 7561 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 7621 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 7681 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 7741 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 7801 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg 7861 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 7921 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 7981 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 8041 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 8101 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taaagcggcc 8161 gcgatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga 8221 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc 8281 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg 8341 tttagagttt ggcaacatat gccatatgct ggctgccatg aacaaaggtg gctataaaga 8401 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg 8461 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta 8521 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc 8581 atagctgtcc ctcttctctt atgaagatcc ctcgacctgc aggaattcga ttacttaagc 8641 ctaggatcca gatctgtaca ttcgaatcga tgaattcgtt aaccatggaa gcttctgcag 8701 cggccgcact agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8761 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 8821 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 8881 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 8941 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 9001 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 9061 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 9121 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 9181 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 9241 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 9301 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 9361 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 9421 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 9481 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 9541 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 9601 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 9661 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 9721 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 9781 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 9841 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 9901 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 9961 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 10021 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 10081 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 10141 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 10201 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 10261 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 10321 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 10381 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 10441 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 10501 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 10561 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 10621 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 10681 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 10741 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 10801 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 10861 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 10921 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 10981 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 11041 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 11101 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 11161 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 11221 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 11281 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 11341 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 11401 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 11461 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 11521 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 11581 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 11641 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 11701 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 11761 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 11821 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 11881 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 11941 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 12001 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 12061 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 12121 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 12181 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 12241 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 12301 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 12361 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 12421 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 12481 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 12541 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 12601 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 12661 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 12721 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 12781 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 12841 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 12901 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 12961 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 13021 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 13081 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 13141 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 13201 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 13261 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 13321 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 13381 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 13441 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 13501 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 13561 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 13621 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 13681 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 13741 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 13801 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 13861 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 13921 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 13981 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 14041 gcggccgcac tagtctagtc tagagcgatc gcccgggttt aaacgctagc caacaagctc 14101 gtcatcgctt tgcagaagag cagagaggat atgctcatcg tctaaagaac tacccatttt 14161 attatatatt agtcacgata tctataacaa gaaaatatat atataataag ttatcacgta 14221 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa 14281 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta 14341 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca 14401 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa 14461 ttttacgcag actatctttc tagggttaaa aaagatttgc gctttactcg acctaaactt 14521 taaacacgtc atagaatctt cgtttgacaa aaaccacatt gtggccaagc tgtgtgacgc 14581 gacgcgcgct aaagaatggc aaaccaagtc gcgcgaggta cccagctttt gttcccttta 14641 gtgagggtta attccgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 14701 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 14761 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 14821 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 14881 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 14941 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 15001 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 15061 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 15121 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 15181 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 15241 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 15301 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 15361 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 15421 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 15481 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 15541 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 15601 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 15661 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 15721 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 15781 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 15841 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 15901 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 15961 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 16021 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 16081 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 16141 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 16201 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 16261 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 16321 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 16381 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 16441 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 16501 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 16561 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 16621 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 16681 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 16741 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 16801 cacatttccc cgaaaagtgc cacctg //