LOCUS Exported 15593 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PiggyBac construct with doxycyline inducible TRE-tight promoter expression of TMP inducible DDdCas9VP192-P300core-p65-HSF1 activator followed by T2A-EGFP as a reporter and IRES-Neo as selection. ACCESSION . VERSION . KEYWORDS PB-tight-DDdCas9VPPH-T2A-GFP-IRES-Neo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15593) AUTHORS Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti EM, Trokovic R, Kere J, Otonkoski T TITLE Human pluripotent reprogramming with CRISPR activators. JOURNAL Nat Commun. 2018 Jul 6;9(1):2643. doi: 10.1038/s41467-018-05067-x. PUBMED 29980666 REFERENCE 2 (bases 1 to 15593) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15593 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 24..128 /gene="bla" /label=AmpR promoter CDS 129..989 /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 complement(347..366) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1160..1748 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1649..1668 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1902..1919 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2036..2057 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2072..2102 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2110..2126 /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)." primer_bind 2134..2150 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2169..2189 /label=T3 /note="T3 promoter, forward primer" promoter 2171..2189 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" repeat_region 2240..2302 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" CDS 2592..2702 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" promoter 2739..3053 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 2747..2765 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2783..2801 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2818..2836 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2854..2872 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2890..2908 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2925..2943 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2961..2979 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 3026..3050 /label=LNCX /note="Human CMV promoter, forward primer" primer_bind 3253..3272 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 3312..3785 /codon_start=1 /gene="folA" /product="E. coli dihydrofolate reductase" /label=ecDHFR /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER" CDS 3813..3839 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 3843..3863 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3873..7973 /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 7977..7997 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8034..8144 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" CDS 8196..8306 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" CDS 8358..8468 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" CDS 8520..8630 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" CDS 8679..10529 /codon_start=1 /gene="EP300" /product="catalytic histone acetyltransferase core of the human E1A-associated protein p300 (Delvecchio et al., 2013)" /label=p300 core /translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD" regulatory 10293..10302 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 10566..11108 /codon_start=1 /product="C-terminal portion of the p65 subunit of mouse NF-kappa-B" /label=p65 /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISS" CDS 11133..11504 /codon_start=1 /product="C-terminal activation domain from the human heat shock transcription factor HSF1" /label=HSF1 /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED PTISLLTGSEPPKAKDPTVS" CDS 11538..11591 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 11598..12317 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 11598..12314 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(11643..11664) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(11904..11923) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 12251..12272 /label=EGFP-C /note="EGFP, forward primer" misc_feature 12339..12912 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(12506..12523) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 12733..12752 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 12926..13720 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(12980..12999) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 13590..13609 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(14022..14039) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 14028..14252 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(14299..14315) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 14323..14339 /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(14347..14377) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14392..14413 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." protein_bind complement(14472..14596) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" repeat_region 14886..14920 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(14949..14968) /label=T7 /note="T7 promoter, forward primer" promoter complement(14950..14968) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(14978..14995) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(14978..14994) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14987..15009) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 15136..15591 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(15223..15242) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 15433..15454 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggattaatta agcggccgca 2221 tacgcgtata ctagattaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa 2281 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat 2341 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa 2401 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata 2461 aagtaacaaa acttttaact agaattgagc tgttcgaccg gcagggagcc gaacctgcct 2521 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg 2581 gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg 2641 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc 2701 tccccgggta actagaattg taaatgcatt caaacgtcga gtttactccc tatcagtgat 2761 agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg agtttactcc 2821 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt 2881 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta tcagtgatag 2941 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga ggtaggcgtg 3001 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagaa 3061 ggatccgcgg ccgcgccggc tctagatcgc gaacgcgtga attgtaaatg cattcaaacg 3121 tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc 3181 acctgtaggt ttggcaagct agagcctctg ctaaccatgt tcatgccttc ttctttttcn 3241 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg 3301 gatccgccac catgatcagt ctgattgcgg cgttagcggt agattacgtt atcggcatgg 3361 aaaacgccat gccgtggaac ctgcctgccg atctcgcctg gtttaaacgc aacaccttaa 3421 ataaacccgt gattatgggc cgccatacct gggaatcaat cggtcgtccg ttgccaggac 3481 gcaaaaatat tatcctcagc agtcaaccga gtacggacga tcgcgtaacg tgggtgaagt 3541 cggtggatga agccatcgcg gcgtgtggtg acgtaccaga aatcatggtg attggcggcg 3601 gtcgcgttat tgaacagttc ttgccaaaag cgcaaaaact gtatctgacg catatcgacg 3661 cagaagtgga aggcgacacc catttcccgg attacgagcc ggatgactgg gaatcggtat 3721 tcagcgaatt ccacgatgct gatgcgcaga actctcacag ctattgcttt gagattctgg 3781 agcggcgagg ctccggaacc ggtgccacca tgtacccata cgatgttcca gattacgctt 3841 cgccgaagaa aaagcgcaag gtcgaagcgt ccgacaagaa gtacagcatc ggcctggcca 3901 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga 3961 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc 4021 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa 4081 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg 4141 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata 4201 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga 4261 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc 4321 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg 4381 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga 4441 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca 4501 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg 4561 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca 4621 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct 4681 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc 4741 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg 4801 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg 4861 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt 4921 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag 4981 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg 5041 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc 5101 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc 5161 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact 5221 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg 5281 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct 5341 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc 5401 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga 5461 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc 5521 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga 5581 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc 5641 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg 5701 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga 5761 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc 5821 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc 5881 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca 5941 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag 6001 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc 6061 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga 6121 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc 6181 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc 6241 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc 6301 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca 6361 accggctgtc cgactacgat gtcgacgcca tcgtgcctca gagctttctg aaggacgact 6421 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc 6481 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc 6541 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac 6601 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg 6661 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc 6721 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc 6781 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg 6841 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg 6901 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca 6961 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta 7021 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg 7081 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc 7141 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc 7201 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt 7261 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa 7321 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa 7381 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga 7441 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga 7501 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca 7561 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg 7621 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg 7681 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc 7741 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc 7801 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca 7861 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga 7921 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacagcccca 7981 agaagaagag aaaggtggag gccagcctcg agtgcgaaag cggcgggccg gccgacgccc 8041 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata 8101 tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg tcactaccca 8161 gcgctagcgt cgagtgcgaa agcggcgggc cggccgacgc ccttgacgat tttgacttag 8221 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc 8281 ttgacgattt tgaccttgac atgctccccg ggtcactacc cagcgctagc gtcgagtgcg 8341 aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc ccagccgatg 8401 cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat tttgaccttg 8461 acatgctccc cgggtcacta cccagcgcta gcgtcgagtg cgaaagcggc gggccggccg 8521 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc 8581 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtcac 8641 tacccagcgc tagcgtcgag ttcgaaggca gcggcgggat tttcaaacca gaagaactac 8701 gacaggcact gatgccaact ttggaggcac tttaccgtca ggatccagaa tcccttccct 8761 ttcgtcaacc tgtggaccct cagcttttag gaatccctga ttactttgat attgtgaaga 8821 gccccatgga tctttctacc attaagagga agttagacac tggacagtat caggagccct 8881 ggcagtatgt cgatgatatt tggcttatgt tcaataatgc ctggttatat aaccggaaaa 8941 catcacgggt atacaaatac tgctccaagc tctctgaggt ctttgaacaa gaaattgacc 9001 cagtgatgca aagccttgga tactgttgtg gcagaaagtt ggagttctct ccacagacac 9061 tgtgttgcta cggcaaacag ttgtgcacaa tacctcgtga tgccacttat tacagttacc 9121 agaacaggta tcatttctgt gagaagtgtt tcaatgagat ccaaggggag agcgtttctt 9181 tgggggatga cccttcccag cctcaaacta caataaataa agaacaattt tccaagagaa 9241 aaaatgacac actggatcct gaactgtttg ttgaatgtac agagtgcgga agaaagatgc 9301 atcagatctg tgtccttcac catgagatca tctggcctgc tggattcgtc tgtgatggct 9361 gtttaaagaa aagtgcacga actaggaaag aaaataagtt ttctgctaaa aggttgccat 9421 ctaccagact tggcaccttt ctagagaatc gtgtgaatga ctttctgagg cgacagaatc 9481 accctgagtc aggagaggtc actgttagag tagttcatgc ttctgacaaa accgtggaag 9541 taaaaccagg catgaaagca aggtttgtgg acagtggaga gatggcagaa tcctttccat 9601 accgaaccaa agccctcttt gcctttgaag aaattgatgg tgttgacctg tgcttctttg 9661 gcatgcatgt tcaagagtat ggctctgact gccctccacc caaccagagg agagtataca 9721 tatcttacct cgatagtgtt catttcttcc gtcctaaatg cttgaggact gcagtctatc 9781 atgaaatcct aattggatat ttagaatatg tcaagaaatt aggttacaca acagggcata 9841 tttgggcatg tccaccaagt gagggagatg attatatctt ccattgccat cctcctgacc 9901 agaagatacc caagcccaag cgactgcagg aatggtacaa aaaaatgctt gacaaggctg 9961 tatcagagcg tattgtccat gactacaagg atatttttaa acaagctact gaagatagat 10021 taacaagtgc aaaggaattg ccttatttcg agggtgattt ctggcccaat gttctggaag 10081 aaagcattaa ggaactggaa caggaggaag aagagagaaa acgagaggaa aacaccagca 10141 atgaaagcac agatgtgacc aagggagaca gcaaaaatgc taaaaagaag aataataaga 10201 aaaccagcaa aaataagagc agcctgagta ggggcaacaa gaagaaaccc gggatgccca 10261 atgtatctaa cgacctctca cagaaactat atgccaccat ggagaagcat aaagaggtct 10321 tctttgtgat ccgcctcatt gctggccctg ctgccaactc cctgcctccc attgttgatc 10381 ctgatcctct catcccctgc gatctgatgg atggtcggga tgcgtttctc acgctggcaa 10441 gggacaagca cctggagttc tcttcactcc gaagagccca gtggtccacc atgtgcatgc 10501 tggtggagct gcacacgcag agccaggacg tcgacactag cgtcgagttc gaaggcagcg 10561 gcgggccttc agggcagatc agcaaccagg ccctggctct ggcccctagc tccgctccag 10621 tgctggccca gactatggtg ccctctagtg ctatggtgcc tctggcccag ccacctgctc 10681 cagcccctgt gctgacccca ggaccacccc agtcactgag cgctccagtg cccaagtcta 10741 cacaggccgg cgaggggact ctgagtgaag ctctgctgca cctgcagttc gacgctgatg 10801 aggacctggg agctctgctg gggaacagca ccgatcccgg agtgttcaca gatctggcct 10861 ccgtggacaa ctctgagttt cagcagctgc tgaatcaggg cgtgtccatg tctcatagta 10921 cagccgaacc aatgctgatg gagtaccccg aagccattac ccggctggtg accggcagcc 10981 agcggccccc cgaccccgct ccaactcccc tgggaaccag cggcctgcct aatgggctgt 11041 ccggagatga agacttctca agcatcgctg atatggactt tagtgccctg ctgtcacaga 11101 tttcctctag tgggcaggga ggaggtggaa gcggcttcag cgtggacacc agtgccctgc 11161 tggacctgtt cagcccctcg gtgaccgtgc ccgacatgag cctgcctgac cttgacagca 11221 gcctggccag tatccaagag ctcctgtctc cccaggagcc ccccaggcct cccgaggcag 11281 agaacagcag cccggattca gggaagcagc tggtgcacta cacagcgcag ccgctgttcc 11341 tgctggaccc cggctccgtg gacaccggga gcaacgacct gccggtgctg tttgagctgg 11401 gagagggctc ctacttctcc gaaggggacg gcttcgccga ggaccccacc atctccctgc 11461 tgacaggctc ggagcctccc aaagccaagg accccactgt ctcctctaga gtcgacacta 11521 gcgtcgacgg ctccggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 11581 atcctggccc aatcgatatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 11641 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 11701 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 11761 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 11821 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 11881 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 11941 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 12001 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 12061 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 12121 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 12181 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 12241 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 12301 acgagctgta caagtaattt aaagaattcc gccccccccc cctctccctc ccccccccct 12361 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt 12421 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg 12481 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc 12541 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt 12601 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta 12661 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg 12721 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag 12781 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag 12841 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa 12901 acacgatgat aagcttgcca caaccatggc tgaacaagat ggattgcacg caggttctcc 12961 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 13021 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 13081 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 13141 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 13201 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 13261 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 13321 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 13381 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 13441 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 13501 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 13561 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 13621 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 13681 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggatcgg ctagagtcga 13741 cccggacggg acgctcctgc gcctgataca gaacgaattg cttgcaggca tctcatgagt 13801 gtgtcttccc gttttccgcc tgaggtcact gcgtggatgg agcgctggcg cctgctgcgc 13861 gacggcgagc tgctcaccac ccactcgagg gctgcagcgc tgcagaggcc gagtgcagaa 13921 ctgctccaaa gggacctcaa ggctttccga gggacactag gctgactcca tcgagccagt 13981 gtagagataa gcttatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag 14041 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 14101 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 14161 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 14221 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa cctgcagccc 14281 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 14341 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 14401 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 14461 ccagcggatc ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt 14521 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt 14581 ttttgtacaa acttgtgcta gcattctagt tgatatctat aacaagaaaa tatatatata 14641 ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct 14701 taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa 14761 aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga 14821 gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa 14881 gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaatctaga atggcgcgcc 14941 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc 15001 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 15061 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 15121 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 15181 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 15241 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 15301 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 15361 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 15421 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 15481 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 15541 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tag //