LOCUS Exported 14236 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses p300-dCas9-VP64 and MS2-p65-HSF1 fusion proteins. ACCESSION . VERSION . KEYWORDS pPB-R1R2_EF1ap300dCas9VP64_T2A_MS2p65HSF1-IRESbsdpA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14236) AUTHORS Chong ZS, Ohnishi S, Yusa K, Wright GJ TITLE Pooled extracellular receptor-ligand interaction screening using CRISPR activation. JOURNAL Genome Biol. 2018 Nov 26;19(1):205. doi: 10.1186/s13059-018-1581-3. PUBMED 30477585 REFERENCE 2 (bases 1 to 14236) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14236 /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 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /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 674..708 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" protein_bind 998..1022 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter 1474..2654 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 1704..2646 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 2603..2623 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 2674..4524 /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 4288..4297 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4558..4605 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4606..8706 /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 N863A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 8761..8781 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8806..8955 /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 8986..9039 /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" regulatory 9046..9055 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 9055..9441 /codon_start=1 /product="bacteriophage MS2 coat protein" /label=MS2-N55K /note="binds with high affinity to a specific stem-loop structure in the viral RNA (Lim et al., 1994)" /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI VKAMQGLLKDGNPIPSAIAANSGIY" CDS 9496..9516 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9532..10074 /codon_start=1 /product="C-terminal portion of the p65 subunit of mouse NF-kappa-B" /label=p65 /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISS" CDS 10099..10470 /codon_start=1 /product="C-terminal activation domain from the human heat shock transcription factor HSF1" /label=HSF1 /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED PTISLLTGSEPPKAKDPTVS" misc_feature 10495..11081 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(10662..10679) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 10889..10908 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 11082..11480 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 11484..11708 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" protein_bind complement(11729..11753) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" repeat_region 11935..11997 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(12048..12068) /label=T3 /note="T3 promoter, forward primer" promoter complement(12048..12066) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(12087..12103) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12087..12103) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12100..12122) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12111..12127 /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(12135..12165) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12180..12201 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12318..12335) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12489..13077) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12569..12588) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13248..14108) /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 13871..13890 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14109..14213) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata 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 gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc 661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat 721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg 781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact 841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta 901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat 961 atattttctt gttatagata tcaactagaa tgctagcaca agtttgtaca aaaaagcagg 1021 ctggcgccgg aaccaattca gtcgactgga tccacgcgtg aggggcgcgg aagccggggc 1081 ggaggaggag ccaaccccgg gctgggctga gacccgcaga ggaagacgct ctagggattt 1141 gtcccggact agcgagatgg caaggctgag gacgggaggc tgattgagag gcgaaggtac 1201 accctaatct caatacaacc tttggagcta agccagcaat ggtagaggga agattctgca 1261 cgtcccttcc aggcggcctc cccgtcacca ccccccccaa cccgccccga ccggagctga 1321 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 1381 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 1441 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 1501 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 1561 agagaaagtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 1621 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 1681 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 1741 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 1801 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 1861 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg 1921 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 1981 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 2041 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 2101 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 2161 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 2221 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 2281 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 2341 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 2401 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 2461 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 2521 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 2581 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 2641 tttcaggtgt cgtggaagct tggggccacc atgattttca aaccagaaga actacgacag 2701 gcactgatgc caactttgga ggcactttac cgtcaggatc cagaatccct tccctttcgt 2761 caacctgtgg accctcagct tttaggaatc cctgattact ttgatattgt gaagagcccc 2821 atggatcttt ctaccattaa gaggaagtta gacactggac agtatcagga gccctggcag 2881 tatgtcgatg atatttggct tatgttcaat aatgcctggt tatataaccg gaaaacatca 2941 cgggtataca aatactgctc caagctctct gaggtctttg aacaagaaat tgacccagtg 3001 atgcaaagcc ttggatactg ttgtggcaga aagttggagt tctctccaca gacactgtgt 3061 tgctacggca aacagttgtg cacaatacct cgtgatgcca cttattacag ttaccagaac 3121 aggtatcatt tctgtgagaa gtgtttcaat gagatccaag gggagagcgt ttctttgggg 3181 gatgaccctt cccagcctca aactacaata aataaagaac aattttccaa gagaaaaaat 3241 gacacactgg atcctgaact gtttgttgaa tgtacagagt gcggaagaaa gatgcatcag 3301 atctgtgtcc ttcaccatga gatcatctgg cctgctggat tcgtctgtga tggctgttta 3361 aagaaaagtg cacgaactag gaaagaaaat aagttttctg ctaaaaggtt gccatctacc 3421 agacttggca cctttctaga gaatcgtgtg aatgactttc tgaggcgaca gaatcaccct 3481 gagtcaggag aggtcactgt tagagtagtt catgcttctg acaaaaccgt ggaagtaaaa 3541 ccaggcatga aagcaaggtt tgtggacagt ggagagatgg cagaatcctt tccataccga 3601 accaaagccc tctttgcctt tgaagaaatt gatggtgttg acctgtgctt ctttggcatg 3661 catgttcaag agtatggctc tgactgccct ccacccaacc agaggagagt atacatatct 3721 tacctcgata gtgttcattt cttccgtcct aaatgcttga ggactgcagt ctatcatgaa 3781 atcctaattg gatatttaga atatgtcaag aaattaggtt acacaacagg gcatatttgg 3841 gcatgtccac caagtgaggg agatgattat atcttccatt gccatcctcc tgaccagaag 3901 atacccaagc ccaagcgact gcaggaatgg tacaaaaaaa tgcttgacaa ggctgtatca 3961 gagcgtattg tccatgacta caaggatatt tttaaacaag ctactgaaga tagattaaca 4021 agtgcaaagg aattgcctta tttcgagggt gatttctggc ccaatgttct ggaagaaagc 4081 attaaggaac tggaacagga ggaagaagag agaaaacgag aggaaaacac cagcaatgaa 4141 agcacagatg tgaccaaggg agacagcaaa aatgctaaaa agaagaataa taagaaaacc 4201 agcaaaaata agagcagcct gagtaggggc aacaagaaga aacccgggat gcccaatgta 4261 tctaacgacc tctcacagaa actatatgcc accatggaga agcataaaga ggtcttcttt 4321 gtgatccgcc tcattgctgg ccctgctgcc aactccctgc ctcccattgt tgatcctgat 4381 cctctcatcc cctgcgatct gatggatggt cgggatgcgt ttctcacgct ggcaagggac 4441 aagcacctgg agttctcttc actccgaaga gcccagtggt ccaccatgtg catgctggtg 4501 gagctgcaca cgcagagcca ggacggaggt ggaagcggag gatgtacggc caccatgaaa 4561 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggacaa gaagtacagc 4621 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 4681 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 4741 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 4801 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 4861 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 4921 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 4981 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 5041 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 5101 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 5161 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 5221 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 5281 gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg 5341 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 5401 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 5461 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 5521 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 5581 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 5641 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 5701 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 5761 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 5821 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 5881 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 5941 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 6001 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 6061 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 6121 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 6181 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 6241 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 6301 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 6361 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 6421 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 6481 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 6541 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 6601 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 6661 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 6721 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 6781 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 6841 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 6901 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 6961 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 7021 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 7081 gaactggaca tcaaccggct gtccgactac gatgtggacc acatcgtgcc tcagagcttt 7141 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaaggc ccggggcaag 7201 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 7261 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 7321 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 7381 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 7441 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 7501 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 7561 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 7621 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 7681 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 7741 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 7801 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 7861 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 7921 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 7981 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 8041 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 8101 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 8161 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 8221 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 8281 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 8341 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 8401 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 8461 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 8521 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 8581 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 8641 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 8701 ggcgacagcg ctggaggagg tggaagcgga ggaggaggaa gcggaggagg aggtagcgga 8761 cctaagaaaa agaggaaggt ggcggccgct ggatccggac gggctgacgc attggacgat 8821 tttgatctgg atatgctggg aagtgacgcc ctcgatgatt ttgaccttga catgcttggt 8881 tcggatgccc ttgatgactt tgacctcgac atgctcggca gtgacgccct tgatgatttc 8941 gacctggaca tgctgattaa ctgtacgggc ggcgggtccg gaggagaggg cagaggaagt 9001 ctcctaacat gcggtgacgt ggaggagaat cctggcccat gtacggccac catggcttca 9061 aactttactc agttcgtgct cgtggacaat ggtgggacag gggatgtgac agtggctcct 9121 tctaatttcg ctaatggggt ggcagagtgg atcagctcca actcacggag ccaggcctac 9181 aaggtgacat gcagcgtcag gcagtctagt gcccagaaga gaaagtatac catcaaggtg 9241 gaggtcccca aagtggctac ccagacagtg ggcggagtcg aactgcctgt cgccgcttgg 9301 aggtcctacc tgaacatgga gctcactatc ccaattttcg ctaccaattc tgactgtgaa 9361 ctcatcgtga aggcaatgca ggggctcctc aaagacggta atcctatccc ttccgccatc 9421 gccgctaact caggtatcta cagcgctgga ggaggtggaa gcggaggagg aggaagcgga 9481 ggaggaggta gcggacctaa gaaaaagagg aaggtggcgg ccgctggatc cccttcaggg 9541 cagatcagca accaggccct ggctctggcc cctagctccg ctccagtgct ggcccagact 9601 atggtgccct ctagtgctat ggtgcctctg gcccagccac ctgctccagc ccctgtgctg 9661 accccaggac caccccagtc actgagcgct ccagtgccca agtctacaca ggccggcgag 9721 gggactctga gtgaagctct gctgcacctg cagttcgacg ctgatgagga cctgggagct 9781 ctgctgggga acagcaccga tcccggagtg ttcacagatc tggcctccgt ggacaactct 9841 gagtttcagc agctgctgaa tcagggcgtg tccatgtctc atagtacagc cgaaccaatg 9901 ctgatggagt accccgaagc cattacccgg ctggtgaccg gcagccagcg gccccccgac 9961 cccgctccaa ctcccctggg aaccagcggc ctgcctaatg ggctgtccgg agatgaagac 10021 ttctcaagca tcgctgatat ggactttagt gccctgctgt cacagatttc ctctagtggg 10081 cagggaggag gtggaagcgg cttcagcgtg gacaccagtg ccctgctgga cctgttcagc 10141 ccctcggtga ccgtgcccga catgagcctg cctgaccttg acagcagcct ggccagtatc 10201 caagagctcc tgtctcccca ggagcccccc aggcctcccg aggcagagaa cagcagcccg 10261 gattcaggga agcagctggt gcactacaca gcgcagccgc tgttcctgct ggaccccggc 10321 tccgtggaca ccgggagcaa cgacctgccg gtgctgtttg agctgggaga gggctcctac 10381 ttctccgaag gggacggctt cgccgaggac cccaccatct ccctgctgac aggctcggag 10441 cctcccaaag ccaaggaccc cactgtctcc tgtacatgag aattccgccc cccccccctc 10501 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 10561 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 10621 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 10681 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 10741 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 10801 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 10861 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct 10921 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg 10981 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt 11041 ggttttcctt tgaaaaacac gatgataata tggccacaac catggccaag cctttgtctc 11101 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg 11161 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca 11221 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg 11281 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct 11341 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca 11401 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg 11461 gttatgtgtg ggagggctga cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 11521 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 11581 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 11641 gcaggacagc aagggggagg attgggaagt caatagcagg catgctgggg atgcggtggg 11701 ctctatggac tagtctcgag atatctagac ccagctttct tgtacaaagt ggtactagtt 11761 aaaagttttg ttactttata gaagaaattt tgagtttttg ttttttttta ataaataaat 11821 aaacataaat aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa 11881 cttaatatct attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg 11941 tcaattttac gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta 12001 atctagtata cgcgtatgcg gccgcttaat taatccagct tttgttccct ttagtgaggg 12061 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 12121 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 12181 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 12241 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 12301 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 12361 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 12421 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 12481 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 12541 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 12601 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 12661 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 12721 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 12781 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 12841 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 12901 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 12961 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 13021 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 13081 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 13141 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 13201 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 13261 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 13321 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 13381 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 13441 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 13501 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 13561 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 13621 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 13681 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 13741 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 13801 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 13861 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 13921 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 13981 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 14041 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 14101 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 14161 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 14221 ccccgaaaag tgccac //