LOCUS Exported 14973 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses human codon-optimized intein-Cas9. ACCESSION . VERSION . KEYWORDS pMK33/intein-Cas9_S219-3XFLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14973) AUTHORS Viswanatha R, Li Z, Hu Y, Perrimon N TITLE Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells. JOURNAL Elife. 2018 Jul 27;7. pii: 36333. doi: 10.7554/eLife.36333. PUBMED 30051818 REFERENCE 2 (bases 1 to 14973) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14973 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(624..641) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(795..1383) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(875..894) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1554..2414) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 2177..2196 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2415..2519) /gene="bla" /label=AmpR promoter primer_bind 2587..2605 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4001..4280 /label=copia promoter /note="strong promoter from the Drosophila transposable element copia (Sinclair et al., 1986)" CDS 4326..5351 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" intron 5714..5779 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" CDS 5909..5929 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 6354..6488 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6391..6410) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6445..6464 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 6489..6915 /label=MT promoter /note="Drosophila metallothionein promoter" primer_bind 6923..6939 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 6924..6940 /label=KS primer /note="common sequencing primer, one of multiple similar variants" protein_bind 6955..6979 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 9955..12339 /codon_start=1 /product="C-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(C) /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD" CDS 12346..12366 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 12373..12438 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 12456..12473 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(12496..12513) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 12502..12726 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" protein_bind complement(12743..12767) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 12790..13536 /codon_start=1 /product="soybean ascorbate peroxidase, engineered to serve as a monomeric tag for electron microscopy and live-cell proteomics (Lam et al., 2015)" /label=APEX2 /note="more active than the original APEX due to the A134P mutation" /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK YAADEDAFFADYAEAHQKLSELGFADA" CDS 13555..13584 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 13588..13617 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 13621..13650 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" ORIGIN 1 cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa 61 ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat cgtcacggcg 121 atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta 181 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga 241 atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat 301 tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg 361 ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc 421 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt 481 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg 541 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc 601 ggaagtcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 661 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 721 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 781 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 841 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 901 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 961 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1021 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1081 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1141 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1201 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 1261 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1321 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1381 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1441 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1501 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 1561 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 1621 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 1681 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 1741 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 1801 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 1861 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 1921 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 1981 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 2041 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 2101 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 2161 ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 2221 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 2281 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 2341 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 2401 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 2461 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 2521 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 2581 tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattcacatt tgtacgaatt 2641 ttttttttat caaaagttcg agtttttcac caatttcctc atcaaccgag caaggcaaac 2701 ggctttgaat aatatggtgt tatatataca tatatcaaat cgctgctgac tgcgtgattg 2761 atggccccaa gattacatat tatcgaatca ggattcagaa ggagatcaat gtcaaatgcg 2821 gacaggaaca tgaaagacgc ctgttatgcg caattaaaaa tttgggttta attgctgtgg 2881 aaactgttgt tggcggcatc ttaagttcct gtttaacaac atcaactact tatgtacgta 2941 gaagcgttta agccatttgc atacagatga gaactggctt ttgtgctaat cagtcaagat 3001 gactccgatg atgatgactc attacctgac cagttttcgc tgctttcttt tcaacaacta 3061 cttgtatatg tattgtatcc aatagcaata cattgaattt ccatggtcta gtcacgtatt 3121 atcatttaat tgacaccaag tcgtgttatt gttgagctat cgagttcagc tcaaacattt 3181 cttattccca tgaataagcc ggcaaaaata tgcaatctat gaaagttaat ataagcaaac 3241 cttactttga ctcaatacca atgcactttg tgtcgatagg ttcacgcaat tgaggcgatt 3301 attccgataa cccaagcgat tgactgttcc cgtttcgatt ccaattgaaa tttggaaatg 3361 tacaatagtt ttgctatatg ctgtcaagta cgctcttatc ttctctgggt tttcttcaga 3421 gtttcgaaac gcttcttctt ttttttgttt tttttttttt ggaatctcgt attttggaag 3481 gggctcccct ctggaatttg ttacactgtc gttatcattg cgaacaagcg gcccgaagct 3541 atcagcgact ttaacattta caatgcactt ttttacgacc aattaaatgt acattttcct 3601 ttcttcgccc gttgataagc gaacgcgatg tggcgcaggc aatgtgttgc tcttgcgaca 3661 caaacgcaat caaaatggat tcaatttcgc tttttcccag tgaaacgaag aacgaaccga 3721 ccatcatgat atgctcctct gcatgttgcg tattgaatca atgacaattt caattaagcc 3781 gcccgttcgt catgcgtttt cgtgcgcttc gaaatgctga taacgctgct gtcctccaac 3841 tgctttgcat gtggacacaa ttccatttat ttaattcttt tatttggatc ggttaaatta 3901 aaaagcgcct tgttacgcat ttaacgttgt ttccggtgcg tggtggtttc atgcttctgg 3961 gaacggcaaa tgggtttagg attgggaacc cctcatcatc tgttggaata tactattcaa 4021 cctacaaaag taacgttaaa caacactact ttatatttga tatgaatggc cacacctttt 4081 atgccataaa acatattgta agagaatacc actcttttta ttccttcttt ccttcttgta 4141 cgttttttgc tgtaagtagg tcgtggtgct ggtgttgcag ttgaaataac ttaaaatata 4201 aatcataaaa ctcaaacata aacttgacta tttatttatt tattaagaaa ggaaatataa 4261 attataaatt acaacaggtt atggacctgc agccaagctt ggcgctcgtc cgggggcaat 4321 gagatatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 4381 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 4441 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 4501 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 4561 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 4621 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 4681 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 4741 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 4801 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 4861 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 4921 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 4981 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 5041 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 5101 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 5161 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 5221 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 5281 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 5341 caaaggaata gagtagatgc cgaccgaaca agagctgatt tcgagaacgc ctcagccagc 5401 aactcgcgcg agcctagcaa ggcaaatgcg agagaacggc cttacgcttg gtggcacagt 5461 tctcgtccac agttcgctaa gctcgctcgg ctgggtcgcg ggagggccgg tcgcagtgat 5521 tcaggccctt ctggattgtg ttggtcccca gggcacgatt gtcatgccca cgcactcggg 5581 tgatctgact gatcccgcag attggagatc gccgcccgtg cctgccgatt gggtgcagat 5641 ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat 5701 ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct 5761 aattgtttgt gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa 5821 tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc 5881 tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa 5941 ggactttcct tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct 6001 tgcttgcttt gctatttaca ccacaaagga aaaagctgca ctgctataca agaaaattat 6061 ggaaaaatat tctgtaacct ttataagtag gcataacagt tataatcata acatactgtt 6121 ttttcttact ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg 6181 tacctttagc tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt 6241 gactagagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc 6301 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt 6361 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 6421 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 6481 tctggatcaa ttcgttgcag gacaggatgt ggtgcccgat gtgactagct ctttgctgca 6541 ggccgtccta tcctctggtt ccgataagag acccagaact ccggcccccc accgcccacc 6601 gccaccccca tacatatgtg gtacgcaagt aagagtgcct gcgcatgccc catgtgcccc 6661 accaagagct ttgcatccca tacaagtccc caaagtggag aaccgaacca attcttcgcg 6721 ggcagaacaa aagcttctgc acacgtctcc actcgaattt ggagccggcc ggcgtgtgca 6781 aaagaggtga atcgaacgaa agacccgtgt gtaaagccgc gtttccaaaa tgtataaaac 6841 cgagagcatc tggccaatgt gcatcagttg tggtcagcag caaaatcaag tgaatcatct 6901 cagtgcaact aaagggggga tctcgaggtc gacggtatcg ataagcttga tatcacaagt 6961 ttgtacaaaa aagcaggctc cgcggccgcc cccttcacca tggataaaaa gtattctatt 7021 ggtttagaca tcggcactaa ttccgttgga tgggctgtca taaccgatga atacaaagta 7081 ccttcaaaga aatttaaggt gttggggaac acagaccgtc attcgattaa aaagaatctt 7141 atcggtgccc tcctattcga tagtggcgaa acggcagagg cgactcgcct gaaacgaacc 7201 gctcggagaa ggtatacacg tcgcaagaac cgaatatgtt acttacaaga aatttttagc 7261 aatgagatgg ccaaagttga cgattctttc tttcaccgtt tggaagagtc cttccttgtc 7321 gaagaggaca agaaacatga acggcacccc atctttggaa acatagtaga tgaggtggca 7381 tatcatgaaa agtacccaac gatttatcac ctcagaaaaa agctagttga ctcaactgat 7441 aaagcggacc tgaggttaat ctacttggct cttgcccata tgataaagtt ccgtgggcac 7501 tttctcattg agggtgatct aaatccggac aactcggatg tcgacaaact gttcatccag 7561 ttagtacaaa cctataatca gttgtttgaa gagaacccta taaatgcaag tggcgtggat 7621 gcgaaggcta ttcttagcgc ccgcctctct aaatgccttg ccgagggtac ccgaatcttc 7681 gatccggtca ctggtacaac gcatcgcatc gaggatgttg tcgatgggcg caagcctatt 7741 catgtcgtgg ctgctgccaa ggacggaacg ctgctcgcgc ggcccgtggt gtcctggttc 7801 gaccagggaa cgcgggatgt gatcgggttg cggatcgccg gtggcgccat cgtgtgggcg 7861 acacccgatc acaaggtgct gacagagtac ggctggcgtg ccgccgggga actccgcaag 7921 ggagacaggg tggccggacc gggtggttct ggtaacagcc tggccttgtc cctgacggcc 7981 gaccagatgg tcagtgcctt gttggatgct gagcccccca tactctattc cgagtatgat 8041 cctaccagtc ccttcagtga agcttcgatg atgggcttac tgaccaacct ggcagacagg 8101 gagctggttc acatgatcaa ctgggcgaag agggtgccag gctttgtgga tttgaccctc 8161 catgatcagg cccaccttct agaacgtgcc tggctagaga tcctgatgat tggtctcgtc 8221 tggcgctcca tggagcaccc agggaagcta ctgtttgctc ctaacttgct cttggacagg 8281 aaccagggaa aatgtgtaga gggcatggtg gagatcttcg acatgctgct ggctacatca 8341 tctcggttcc gcatgatgaa tctgcaggga gaggagtttg tgtgcctcaa atctattatt 8401 ttgcttaatt ctggagtgta cacatttctg tccagcaccc tgaagtctct ggaagagaag 8461 gaccatatcc accgagccct ggacaagatc acggacactt tgatccacct gatggccaag 8521 gcaggcctga ccctgcagca gcagcaccag cggctggccc agctcctcct catcctctcc 8581 cacatcaggc acatgagtaa caaaggaatg gagcatctgt acagcatgaa gtacaagaac 8641 gtggtgcccc tctatgacct gctgctggag atgctggacg cccaccgcct acatgcgggt 8701 ggttctggtg ctagccgcgt gcaggcgttc gcggatgccc tggatgacaa attcctgcac 8761 gacatgctgg cggaaggact ccgctattcc gtgatccgag aagtgctgcc aacgcggcgg 8821 gcacgaacgt tcgacctcga ggtcgaggaa ctgcacaccc tcgtcgccga aggggttgtc 8881 gtgcacaact gccgacggct agaaaacctg atcgcacaat tacccggaga gaagaaaaat 8941 gggttgttcg gtaaccttat agcgctctca ctaggcctga caccaaattt taagtcgaac 9001 ttcgacttag ctgaagatgc caaattgcag cttagtaagg acacgtacga tgacgatctc 9061 gacaatctac tggcacaaat tggagatcag tatgcggact tatttttggc tgccaaaaac 9121 cttagcgatg caatcctcct atctgacata ctgagagtta atactgagat taccaaggcg 9181 ccgttatccg cttcaatgat caaaaggtac gatgaacatc accaagactt gacacttctc 9241 aaggccctag tccgtcagca actgcctgag aaatataagg aaatattctt tgatcagtcg 9301 aaaaacgggt acgcaggtta tattgacggc ggagcgagtc aagaggaatt ctacaagttt 9361 atcaaaccca tattagagaa gatggatggg acggaagagt tgcttgtaaa actcaatcgc 9421 gaagatctac tgcgaaagca gcggactttc gacaacggta gcattccaca tcaaatccac 9481 ttaggcgaat tgcatgctat acttagaagg caggaggatt tttatccgtt cctcaaagac 9541 aatcgtgaaa agattgagaa aatcctaacc tttcgcatac cttactatgt gggacccctg 9601 gcccgaggga actctcggtt cgcatggatg acaagaaagt ccgaagaaac gattactcca 9661 tggaattttg aggaagttgt cgataaaggt gcgtcagctc aatcgttcat cgagaggatg 9721 accaactttg acaagaattt accgaacgaa aaagtattgc ctaagcacag tttactttac 9781 gagtatttca cagtgtacaa tgaactcacg aaagttaagt atgtcactga gggcatgcgt 9841 aaacccgcct ttctaagcgg agaacagaag aaagcaatag tagatctgtt attcaagacc 9901 aaccgcaaag tgacagttaa gcaattgaaa gaggactact ttaagaaaat tgaatgcttc 9961 gattctgtcg agatctccgg ggtagaagat cgatttaatg cgtcacttgg tacgtatcat 10021 gacctcctaa agataattaa agataaggac ttcctggata acgaagagaa tgaagatatc 10081 ttagaagata tagtgttgac tcttaccctc tttgaagatc gggaaatgat tgaggaaaga 10141 ctaaaaacat acgctcacct gttcgacgat aaggttatga aacagttaaa gaggcgtcgc 10201 tatacgggct ggggacgatt gtcgcggaaa cttatcaacg ggataagaga caagcaaagt 10261 ggtaaaacta ttctcgattt tctaaagagc gacggcttcg ccaataggaa ctttatgcag 10321 ctgatccatg atgactcttt aaccttcaaa gaggatatac aaaaggcaca ggtttccgga 10381 caaggggact cattgcacga acatattgcg aatcttgctg gttcgccagc catcaaaaag 10441 ggcatactcc agacagtcaa agtagtggat gagctagtta aggtcatggg acgtcacaaa 10501 ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa ggggcaaaaa 10561 aacagtcgag agcggatgaa gagaatagaa gagggtatta aagaactggg cagccagatc 10621 ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg agaaacttta cctctattac 10681 ctacaaaatg gaagggacat gtatgttgat caggaactgg acataaaccg tttatctgat 10741 tacgacgtcg atcacattgt accccaatcc tttttgaagg acgattcaat cgacaataaa 10801 gtgcttacac gctcggataa gaaccgaggg aaaagtgaca atgttccaag cgaggaagtc 10861 gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg cgaaactgat aacgcaaaga 10921 aagttcgata acttaactaa agctgagagg ggtggcttgt ctgaacttga caaggccgga 10981 tttattaaac gtcagctcgt ggaaacccgc caaatcacaa agcatgttgc acagatacta 11041 gattcccgaa tgaatacgaa atacgacgag aacgataagc tgattcggga agtcaaagta 11101 atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt ctataaagtt 11161 agggagataa ataactacca ccatgcgcac gacgcttatc ttaatgccgt cgtagggacc 11221 gcactcatta agaaataccc gaagctagaa agtgagtttg tgtatggtga ttacaaagtt 11281 tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc tacagccaaa 11341 tacttctttt attctaacat tatgaatttc tttaagacgg aaatcactct ggcaaacgga 11401 gagatacgca aacgaccttt aattgaaacc aatggggaga caggtgaaat cgtatgggat 11461 aagggccggg acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt caacatagta 11521 aagaaaactg aggtgcagac cggagggttt tcaaaggaat cgattcttcc aaaaaggaat 11581 agtgataagc tcatcgctcg taaaaaggac tgggacccga aaaagtacgg tggcttcgat 11641 agccctacag ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg aaaatccaag 11701 aaactgaagt cagtcaaaga attattgggg ataacgatta tggagcgctc gtcttttgaa 11761 aagaacccca tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa ggatctcata 11821 attaaactac caaagtatag tctgtttgag ttagaaaatg gccgaaaacg gatgttggct 11881 agcgccggag agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata cgtgaatttc 11941 ctgtatttag cgtcccatta cgagaagttg aaaggttcac ctgaagataa cgaacagaag 12001 caactttttg ttgagcagca caaacattat ctcgacgaaa tcatagagca aatttcggaa 12061 ttcagtaaga gagtcatcct agctgatgcc aatctggaca aagtattaag cgcatacaac 12121 aagcacaggg ataaacccat acgtgagcag gcggaaaata ttatccattt gtttactctt 12181 accaacctcg gcgctccagc cgcattcaag tattttgaca caacgataga tcgcaaacga 12241 tacacttcta ccaaggaggt gctagacgcg acactgattc accaatccat cacgggatta 12301 tatgaaactc ggatagattt gtcacagctt gggggtgacg gatcccccaa gaagaagagg 12361 aaagtctcga gcgactacaa agaccatgac ggtgattata aagatcatga catcgattac 12421 aaggatgacg atgacaaggc tgcaggatga ccggtcatca tcaccatcac cattgagttt 12481 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 12541 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 12601 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 12661 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 12721 ctatggaagg gtgggcgcgc cgacccagct ttcttgtaca aagtggtgat atccggtggc 12781 ggaggcagtg gaaagtctta cccaactgtg agtgctgatt accaggacgc cgttgagaag 12841 gcgaagaaga agctcagagg cttcatcgct gagaagagat gcgctcctct aatgctccgt 12901 ttggcattcc actctgctgg aacctttgac aagggcacga agaccggtgg acccttcgga 12961 accatcaagc accctgccga actggctcac agcgctaaca acggtcttga catcgctgtt 13021 aggcttttgg agccactcaa ggcggagttc cctattttga gctacgccga tttctaccag 13081 ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta aggttccatt ccaccctgga 13141 agagaggaca agcctgagcc accaccagag ggtcgcttgc ccgatcccac taagggttct 13201 gaccatttga gagatgtgtt tggcaaagct atggggctta ctgaccaaga tatcgttgct 13261 ctatctgggg gtcacactat tggagctgca cacaaggagc gttctggatt tgagggtccc 13321 tggacctcta atcctcttat tttcgacaac tcatacttca cggagttgtt gagtggtgag 13381 aaggaaggtc tccttcagct accttctgac aaggctcttt tgtctgaccc tgtattccgc 13441 cctctcgttg acaaatatgc agcggacgaa gatgccttct ttgctgatta cgctgaggct 13501 caccaaaagc tttccgagct tgggtttgct gatgccagcg gaggcggtgg ctccgaacag 13561 aaacttatca gcgaggagga tctcggcgag cagaagctga tttcggaaga agaccttgga 13621 gagcaaaagc tcatatccga agaggacctg tcgtaagaat tcctgcagcc cgggggatcc 13681 actagttcta gtcgaccatg aagatcaaga tcattgcccc gccagagcgc aagtactctg 13741 tctggatcgg tggctccatc ctggcttcgc tgtccacctt ccagcagatg tggatctcca 13801 agcaggagta cgacgagtcc ggcccctcca ttgtgcaccg caagtgcttc taagaaggat 13861 cgcttgtctg ggcaagagga tcaggatcgg gatggtcttg attctgctgg aggaggagga 13921 ggagaagtcg aggaagcagc agcgaaagtg caagtgcgag tggtggaagt ttggagtgca 13981 gcacaacaaa atcaacaaca acaccaacta caagatgaaa agagcggaac cacctgcaca 14041 ccatcatcac tatcatcatc gttttgggcg catgttgtgt ggttccagcg tattaatata 14101 attaatttat tccacatgag atatgatatg atatactatg tattttttgt ttttttttta 14161 tttgtaaacc tttaatataa caagaactac aaaaaatgaa aatgagcgaa aatgcatatt 14221 ctgccattcc acacacacac caacaacacc caacacacgc acacccacaa gcttacacac 14281 acacattcgc ggcatgacaa ggacatcaag ataaagaaga acttaaagaa gatatttccc 14341 aaagcgcaaa aagaacacac acacattgca aaacacaaac aacacactag cgttttgtac 14401 aattcgtcag caaccttatg tattattttt taattatgat gtaattataa acaaagtgaa 14461 acaaaaatat gaaaacaaaa aggaaaatca aatctgtctt ctctttctcc cgctctcctc 14521 gctctctgct gctaacctcg ccctctcctc tctcatcttt ttgtctgtct ctcttccaca 14581 tttttgccgg ccggcaaaat aataacccac acacactcac acttggctgc agtttcgcgt 14641 gcgatattca cacacattca agcatacaag catacataca tatgtatttt ttttttattt 14701 gtacactttt ctaattgcat gcgtatcgat tgataagttt acgcctgaaa atgttaatta 14761 aaatgtgaaa atgcaactga aaaactgatg aaatgaaaca acaacaagcg aacaatttgc 14821 tacatgtgta ttgtctaaca accgttactg caacggttgc ttcggaaaag ggtgaagagg 14881 aagagggcac gaggtcgact agagcggccg ccgacgcgag gctggatggc cttccccatt 14941 atgattcttc tcgcttccgg cggcatcggg atg //