LOCUS Exported 13808 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION SP-dCas9-VPR with doxycycline-inducible expression. ACCESSION . VERSION . KEYWORDS PB-TRE-dCas9-VPR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13808) AUTHORS Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM TITLE Highly efficient Cas9-mediated transcriptional programming. JOURNAL Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312. PUBMED 25730490 REFERENCE 2 (bases 1 to 13808) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13808 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(1..105) /gene="bla" /label=AmpR promoter rep_origin complement(131..586) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(268..289) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 480..499 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 713..735 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 727..744 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 728..744 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 874..936 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter 1386..1764 /label=TRE3G promoter /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G" protein_bind 1394..1412 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1430..1448 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1466..1484 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1502..1520 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1538..1556 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1574..1592 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1610..1628 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 1673..1697 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 1791..1800 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1797..5900 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9m4 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the mutations D10A, D839A, H840A, and N863A (Mali et al., 2013)" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5913..5933 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5958..6107 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 6132..6152 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6582..6938 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" CDS 6957..7526 /codon_start=1 /product="transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/BRLF1 (Hardwick et al., 1992; Chavez et al., 2015)" /label=Rta AD /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL DTFLNDECLLHAMHISTGLSIFDTSLF" primer_bind complement(7581..7600) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 7625..7673 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" promoter 7904..8115 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 8128..8396 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" CDS 8430..9173 /codon_start=1 /product="modified rtTA protein that binds tightly to promoters containing the tet operator in the presence of doxycycline" /label=Tet-On(R) 3G /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" CDS 9174..9227 /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 9249..10268 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE" primer_bind complement(10325..10345) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 10379..10500 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(10416..10435) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 10470..10489 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 11262..11296 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(11787..11803) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11787..11803) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11800..11822) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11811..11827 /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(11835..11865) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11880..11901 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12018..12035) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12189..12777) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12269..12288) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12948..13808) /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 13571..13590 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 901 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca 1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca 1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct 1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag 1321 acacctgggg ggatacgggg aaaaggcctc cacggccact agtctttcgt cttcaagaat 1381 tcctcgagtt tactccctat cagtgataga gaacgtatga agagtttact ccctatcagt 1441 gatagagaac gtatgcagac tttactccct atcagtgata gagaacgtat aaggagttta 1501 ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga tagagaacgt 1561 atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact ccctatcagt 1621 gatagagaac gtataagctt taggcgtgta cggtgggcgc ctataaaagc agagctcgtt 1681 tagtgaaccg tcagatcgcc tggagcaatt ccacaacact tttgtcttat accaactttc 1741 cgtaccactt cctaccctcg taaaggtcta gagctagcga attcgaattt gccaccatgg 1801 acaagaagta ctccattggg ctcgctatcg gcacaaacag cgtcggctgg gccgtcatta 1861 cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc gatcgccaca 1921 gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg gccgaagcca 1981 cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg atctgctacc 2041 tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc cataggctgg 2101 aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc tttggcaata 2161 tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg aggaagaagc 2221 ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg gcgcatatga 2281 tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac agcgatgtcg 2341 acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag aacccgatca 2401 acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc 2461 tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt ggtaatctta 2521 tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg gccgaagatg 2581 ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg ctggcccaga 2641 tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac gccattctgc 2701 tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc gctagtatga 2761 tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt gtcagacagc 2821 aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc tacgccggat 2881 acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc atcttggaaa 2941 aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg ttgcgcaaac 3001 agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa ctgcacgcta 3061 tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa aagattgaga 3121 aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga aattccagat 3181 tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc gaggaagtcg 3241 tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt gataaaaatc 3301 tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc acagtttata 3361 acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca ttcctgtctg 3421 gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa gttaccgtga 3481 aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg 3541 gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg aaaatcatta 3601 aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac attgtcctca 3661 cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact tacgctcatc 3721 tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga tgggggcggc 3781 tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca atcctggatt 3841 ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat gatgactctc 3901 tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac agtcttcacg 3961 agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg cagaccgtta 4021 aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat atcgttatcg 4081 agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg gaaaggatga 4141 agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa cacccagttg 4201 aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac ggcagggaca 4261 tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg gctgctatcg 4321 tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca agatccgata 4381 aagctagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt 4441 attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat aatctgacta 4501 aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa aggcagcttg 4561 ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc atgaacacca 4621 agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg aagtctaagc 4681 tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc aacaattacc 4741 accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc 4801 ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt aggaaaatga 4861 tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt tacagcaata 4921 ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg aagcgaccac 4981 ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg gatttcgcga 5041 cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc gaagtacaga 5101 ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac 5161 gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca gtcgcttaca 5221 gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg 5281 aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc atcgactttc 5341 tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt cccaagtact 5401 ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga 5461 aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg gccagccact 5521 atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc gtggaacaac 5581 acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa agagtgatcc 5641 tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg gataagccca 5701 tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg ggcgcgcctg 5761 cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct acaaaggagg 5821 tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca agaatcgacc 5881 tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag gtggaggcca 5941 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg 6001 ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg 6061 acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa 6121 gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc gacaccgacg 6181 accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag agcatcatga 6241 agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga atcgccgtgc 6301 ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc ttcaccagca 6361 gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc ggccagatct 6421 ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct cctgctcctg 6481 caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg cctgtgctgg 6541 ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag gccggcgagg 6601 gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg ggagccctgc 6661 tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac aacagcgagt 6721 tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag cccatgctga 6781 tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct cctgatccag 6841 ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc gacgaggact 6901 tcagctctat cgccgacatg gacttctccg cactgctggg tagcggatcg ggatctcggg 6961 attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt 7021 ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct ccaggaagtc 7081 catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt ccagtacatg 7141 agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca gcgcccgcag 7201 tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc caggctgtca 7261 aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct gcaatctgtg 7321 gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac 7381 ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga 7441 ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac 7501 tgtccatctt cgacacatct ctgttttgaa agggttcgat ccctaccggt tagtaatgag 7561 tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct 7621 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg 7681 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca 7741 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg 7801 gctcgcagcc aacgtcgggg cggcaggccc tgccatagca gatctgcgaa atcggatcca 7861 gctaaatcta agtggatctg cgatcgctcc ggtgcccgtc agtgggcaga gcgcacatcg 7921 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc ctagagaagg 7981 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt 8041 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt 8101 gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg cgcccgccgc 8161 cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc cgcctgtggt 8221 gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag accgggcctt 8281 tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct ttgcctgacc 8341 ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta cagatccaag 8401 ctgtgaccgg cgcctactct agagccgcca tgtctagact ggacaagagc aaagtcataa 8461 actctgctct ggaattactc aatggagtcg gtatcgaagg cctgacgaca aggaaactcg 8521 ctcaaaagct gggagttgag cagcctaccc tgtactggca cgtgaagaac aagcgggccc 8581 tgctcgatgc cctgccaatc gagatgctgg acaggcatca tacccactcc tgccccctgg 8641 aaggcgagtc atggcaagac tttctgcgga acaacgccaa gtcataccgc tgtgctctcc 8701 tctcacatcg cgacggggct aaagtgcatc tcggcacccg cccaacagag aaacagtacg 8761 aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg cttctccctg gagaacgcac 8821 tgtacgctct gtccgccgtg ggccacttta cactgggctg cgtattggag gaacaggagc 8881 atcaagtagc aaaagaggaa agagagacac ctaccaccga ttctatgccc ccacttctga 8941 aacaagcaat tgagctgttc gaccggcagg gagccgaacc tgccttcctt ttcggcctgg 9001 aactaatcat atgtggcctg gagaaacagc taaagtgcga aagcggcggg ccgaccgacg 9061 cccttgacga ttttgactta gacatgctcc cagccgatgc ccttgacgac tttgaccttg 9121 atatgctgcc tgctgacgct cttgacgatt ttgaccttga catgctcccc ggggagggca 9181 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctgaa ttcatggata 9241 gatccggaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg 9301 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg 9361 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag 9421 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca 9481 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt 9541 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg 9601 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag 9661 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt 9721 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg 9781 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg 9841 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg 9901 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg 9961 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc 10021 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg 10081 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg 10141 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg 10201 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa 10261 aggaataggc caaggaacct ctggattaca aaatttgtga aagattgact ggtattctta 10321 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcagttaa 10381 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 10441 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 10501 tcatgtctgg aattgactca aatgatgtca attagtctat cagaagctat ctggtctccc 10561 ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca aactgggttc 10621 ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt 10681 ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc cttttttagg 10741 gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc 10801 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc 10861 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc 10921 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc 10981 tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca 11041 tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca 11101 tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga 11161 caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg 11221 attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca 11281 gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt cttttttccg 11341 gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag cccgtgcctt 11401 ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct gctgcattga 11461 cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt 11521 taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc ttttccggac 11581 acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg gcgacagccg 11641 gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat attacgtcag 11701 cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc gtgagttacc 11761 cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 11821 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 11881 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 11941 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 12001 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 12061 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 12121 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 12181 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 12241 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 12301 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 12361 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 12421 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 12481 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 12541 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 12601 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 12661 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 12721 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 12781 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 12841 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 12901 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 12961 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 13021 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 13081 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 13141 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 13201 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 13261 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 13321 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 13381 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 13441 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 13501 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 13561 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 13621 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 13681 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 13741 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 13801 atactcat //