LOCUS Exported 16349 bp ds-DNA circular SYN 30-AUG-2016 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pT2K U6 stuffer dCas9 VP160 T2A (no puro) IRES mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16349) AUTHORS Tumor Cell Biology TITLE Direct Submission JOURNAL Exported Jul 24, 2018 from SnapGene Viewer 4.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16349 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(7..462) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 604..620 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 630..648 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 3068..3110 /label=Fragment.REV enhancer 3081..3082 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" misc_RNA complement(3127..3202) /label=gRNA scaffold /note="guide RNA scaffold for the CRISPR/Cas9 system" promoter complement(5092..5332) /label=U6 Promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(5303..5347) /label=Fragment.FOR enhancer 5335..5714 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5717..5992 /label=chicken beta-actin promoter primer_bind 6958..6987 /label=pT2K-CAGGS insertion detection F CDS 7075..7101 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 7105..7125 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7135..11235 /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 D10 mutation in the RuvC-like domain and the H840A mutation in the HNH 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 11239..11259 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" misc_feature 11332..11481 /label=VP64 misc_feature 11488..11637 /label=VP64 CDS 11701..11754 /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" misc_feature 11778..12339 /label=IRES /note="IRES from MSCV iresGFP. Last 6 bp are not from EMCV, and are unique to this vector construction." primer_bind complement(11814..11833) /label=SJVL-366_Max=871 /note="in IRES facing left. Max recorded sequencing read = 871 bases. Perfect match Tm = 57.2 C, although for oligos less than 35 bp, only the 3 prime 17 nt must match to trigger annotation. The actual sequence of the primer is CAAACGCACA CCGGCCTTAT . This primer matches this site completely." primer_bind 12198..12219 /label=SJVL-367_Max= /note="in IRES facing right. Max recorded sequencing read = bases. Perfect match Tm = 59.1 C, although for oligos less than 35 bp, only the 3 prime 17 nt must match to trigger annotation. The actual sequence of the primer is TGCCCAGAAG GTACCCCATT GT. This primer matches this site perfectly." CDS 12340..13050 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry corrected snapgene /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(13051..13061) /label=SJVL-749_Max= /note="Venus YFP pcr right. Max recorded sequencing read = bases. Perfect match Tm = 79.9 C, although for oligos less than 35 bp, only the 3 prime 17 nt must match to trigger annotation. The actual sequence of the primer is GCGGCCGCTC ACTTGTACAG CTCGTCCATG CCG. This primer does not match this site completely." polyA_site 13156..13211 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal" primer_bind complement(14112..14128) /label=SK primer /note="common sequencing primer, one of multiple similar variants" promoter complement(14165..14183) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(14204..14220) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 14228..14244 /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(14252..14282) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14297..14318 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(14606..15194) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(15365..16225) /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" promoter complement(16226..16330) /gene="bla" /label=AmpR promoter ORIGIN 1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc 481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 661 ccaaatagta ggaattaccc acctgtacaa gtgctgaaaa cttggatgaa taagcccgtt 721 tgccattttc tactgctatt ttaaatcttt tctgtatttg tctgcatttg tctttaccct 781 tgaataaatg ttttagttgt tttttttcaa ttatgactgt gtttaacaga cattattaca 841 ataatatgta atagtagtac acactattat tatgtaatag tacttgttga ctgtatttga 901 gaactggacc gagtgagtgt tacgtcaccc attcaaaatg acttacttct ggctccaaca 961 aaatgaagtt aattcagttg ccatttttca ctgtatggac atcgccgtgt tggagctaga 1021 cgtcgccaat aagcaagaaa gagccgagat gcgtcgagtc tgagtcaccg ttcctatggc 1081 aacccctcta accaatcaga agtaagcttg ttggaagtcc acagcctacc acttgaaagc 1141 gggctgcaca aaatctgtca aacgttttga acgttggatg tgagagcaca tacttttatt 1201 aaggcatctg gttggtcagt ttataatatc aacaacttgg gctacagaaa agaaaagtta 1261 ttacagaaat tatgattaac aagtacatgt taaataaaga ttttaatatg aatgccacca 1321 ctggagcatt catgccattt ggagcttctt cctgtttgga tcactagaag gaggaggtca 1381 ctcattacag ttctcatata cagtcgttgg ttggttggtt ggttggttgg ttggtagatt 1441 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt 1501 gattgattga ttgattgatt gattgattga ttgattgatt ggtagtcaaa ataagaaata 1561 atttccacag attcattaca gaaatgatta aatgcataca taaaaaactg gggggggggg 1621 gatacaacaa cacacttaag tcacatttgc ctacgtaaag aaaagtaaag aaaatcaata 1681 gctatatttt acatctcctt tttttgctgt cttttaatta gccttgtttt gctgttatct 1741 tgattgaaac tgtaacttct tcacctgctt cttttctttg taggttttgc cagcccagaa 1801 acaggcatgg ctgtgcaagg ccagagcacc atgcacaatg cgctgcatgt cttcatgaac 1861 ggctcaatgt cctcagtcca gggctcagcc aacgacccca tcttcctcct tcaccatgct 1921 ttcattgaca ggtaacaaac acgtcatgac attagactgc acagtttttg acaaagttca 1981 tacaatctgt tgtttatagc tgctacaatt agtgaagttt gtgaatgtac ttggatgagc 2041 agcgaaagat caattgagat caattgttag agtttggttg ccctgcagag caaagaacaa 2101 aaaataatct ggtggcttta ctgcgtgagg ttattattgg tggaatagaa acacaaaaca 2161 taattgcatt tatttgttta attttttatc ttatcttaac tttcatcttg catatttgtt 2221 tcttacatca tttctagcat ctttgagcgc tggctaagaa ctcatcagcc tccccggtcc 2281 atctacccac gtaccaatgc accaattggc cacaatgacg gctactacat ggtgccattc 2341 cttcctcttt ataggaatgg agactacctc ctgtccaaca aggctcttgg atacgagtac 2401 gcctacctgt tggacccagg tcattgcaca acaccagaaa tgccctctga tctgcaaaag 2461 acgtgaatat ctgttcagac acccatatcc actctgttcc acacaggtca gaggtttgtc 2521 caggagttct tgacagaggt gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa 2581 gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa tcttacttga 2641 gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag taaaagtaaa 2701 agcaagaaag aaaactagag attcttgttt aagcttttaa tctcaaaaaa cattaaatga 2761 aatgcataca aggttttatc ctgctttaga actgtttgta tttaattatc aaactataag 2821 acagacaatc taatgccagt acacgctact caaagttgta aaacctcaga tttaacttca 2881 gtagaagctg attctcaaaa ttgttagtgt caagcctagc tcttttgggg ctgaaaagca 2941 atcctgcagt gctgaaaagc ctctcacagg cagccgatgc gggaagaggt gtattagtct 3001 tgatagagag gctgcaaata gcaggaaacg tgagcagaga ctccctggtg tctgaaacac 3061 aggccagatg ggccctcgtc gaccaattcc cactcctttc aagacctagc tagcgaattc 3121 aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac 3181 ttgctatttc tagctctaaa acagagacgt acaaaaaaga gcaagaagct aaaaaagatt 3241 taaaaattat ttttagcgca gttaatggaa caggaactaa atttacccca aaaatattac 3301 gtgaatcagg atataacgtt attgaggttg aagagcatgc atttgaagat gaaacattta 3361 aaaatgttgt aaatccaaat ccagaatttg atcctgcatg aaaaataccg cttgaatatg 3421 gtattaaaca tgatgcagat attattatta tgaatgaccc agatgctgac agatttggaa 3481 tggcaataaa acatgatggt cattttgtaa gattagatgg aaatcaaaca ggaccaattt 3541 taattgattg aaaattatca aatctaaaac gcttaaatag cattccaaaa aatccggctc 3601 tatattcaag ttttgtaaca agtgatttgg gtgatagaat cgctcatgaa aaatatggag 3661 ttaatattgt aaaaacttta actggattta aatgaatggg tagagaaatt gctaaagaag 3721 aagataacgg attaaatttt gtttttgctt atgaagaaag ttatggatat gtaattgatg 3781 actcagctag agataaagat ggaatacaag cttctatatt aatagcagag gctgcttgat 3841 tttataaaaa acaaaataaa acattagtag actatttaga agatttattt aaagaaatgg 3901 gtgcatatta cactttcact ttaaacttga attttaaacc agaagaaaag aaattaaaaa 3961 ttgaaccatt aatgaaatca ttgagagcaa cacccttaac tcaaattgct ggacttaaag 4021 ttgttaatgt tgaagactac atcgatggaa tgtataatat gccaggacaa gacttactaa 4081 aattttattt agaagataag tcatgatttg ctgttcgccc aagtggaact gaacctaaac 4141 taaaaattta ttttataggt gttggtgaat ctgttcaaaa cgctaaagtt aaagtagacg 4201 aaattattaa agaattaaaa ttaaaaatga atatatagga gaaaaaatga aactaaacaa 4261 atatatagat cacacattat taaaacaaga tgctacgaaa gctgaaatta aacaattatg 4321 tgatgaagca attgaatttg attttgcaac agtttgtgtt aattcatatt gaacaagcta 4381 ttgtaaagaa ttattaaaag gcacaaatgt aggaataaca aatgttgtag gttttcctct 4441 aggtgcatgc acaacagcta caaaagcatt cgaagtttct gaagcaatta aagatggtgc 4501 aacagaaatt gatatggtat taaatattgg tgcattaaaa gacaaaaatt atgaattagt 4561 tttagaagac atgaaagctg taaaaaaagc agctggatca catgttgtta aatgtattat 4621 ggaaaattgt ttattaacaa aagaagaaat catgaaagct tgtgaaatag ctgttgaagc 4681 tggattagaa tttgttaaaa catcaacagg attttcaaaa tcaggtgcaa catttgaaga 4741 tgttaaacta atgaagtcag ttgttaaaga caatgcttta gttaaagcag ctggtggagt 4801 tagaacattt gaagatgctc aaaaaatgat tgaagcagga gctgaccgct taggaacaag 4861 tggtggagta gctattatta aaggtgaaga aaacaacgcg agttactaaa actagcgttt 4921 ttttattttg ctcattttta ttaaaagttt gcaaaaagga acataaaaat tctaattatt 4981 gatactaaag ttattaaaaa gaagattttg gttgatttta taaaggtcat agaatataat 5041 attttagcat gtgtattttg tgtgctcatt tacaaccgtc tccggtgttt cgtcctttcc 5101 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt 5161 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc 5221 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa 5281 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tctcgacatt 5341 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 5401 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 5461 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 5521 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 5581 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 5641 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 5701 tcgctattac catgggtcga ggtgagcccc acgttctgct tcactctccc catctccccc 5761 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg 5821 gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 5881 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 5941 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 6001 tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg 6061 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa 6121 ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa gccttaaagg 6181 gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc gtgtgtgtgt 6241 gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct gcgggcgcgg 6301 cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg cggtgccccg 6361 cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt 6421 gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga 6481 gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg cgcggggctc 6541 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgc gcggggcggg gccgcctcgg 6601 gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc tgtcgaggcg 6661 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt 6721 tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg 6781 cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg 6841 ccgcgccgcc gtccccttct ccatctccag cctcggggct gccgcagggg gacggctgcc 6901 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagct 6961 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg 7021 ctggttgttg tgctgtctca tcattttggc aaagaattca ccggtgccac catgtaccca 7081 tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag 7141 aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac 7201 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 7261 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 7321 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 7381 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 7441 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 7501 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 7561 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 7621 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 7681 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 7741 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 7801 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc 7861 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 7921 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 7981 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 8041 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 8101 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 8161 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 8221 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 8281 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 8341 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 8401 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 8461 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 8521 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 8581 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 8641 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 8701 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 8761 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 8821 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 8881 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 8941 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 9001 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 9061 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 9121 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 9181 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 9241 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 9301 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 9361 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 9421 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 9481 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 9541 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 9601 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc catcgtgcct 9661 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 9721 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 9781 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 9841 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 9901 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 9961 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 10021 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 10081 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 10141 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 10201 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 10261 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 10321 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 10381 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 10441 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 10501 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 10561 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 10621 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 10681 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 10741 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 10801 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 10861 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 10921 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 10981 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 11041 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 11101 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 11161 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 11221 cagctgggag gcgacagccc caagaagaag agaaaggtgg aggccagcgg gccggccgga 11281 tccgggcgcg ccgacgcgct ggacgatttc gatctcgaca tgctgggttc tgatgccctc 11341 gatgactttg acctggatat gttgggaagc gacgcattgg atgactttga tctggacatg 11401 ctcggctccg atgctctgga cgatttcgat ctcgatatgt tagggtcaga cgcactggat 11461 gatttcgacc ttgatatgtt gggaagcgat gcccttgatg atttcgacct ggacatgctc 11521 ggcagcgacg ccctggacga tttcgatctg gacatgctgg ggtccgatgc cttggatgat 11581 tttgacttgg atatgctggg gagtgatgcc ctggacgact ttgacctgga catgctgggc 11641 tccgatgcgc tcgatgactt cgatttggat atgttgtata tcgatagatc tggcagcgga 11701 gagggcagag gaagccttct aacatgcggt gacgtggagg agaatcccgg ccctaggctc 11761 gagcgggatc aattccgccc cccccctaac gttactggcc gaagccgctt ggaataaggc 11821 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg 11881 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc 11941 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga 12001 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg 12061 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag 12121 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc 12181 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct 12241 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa acgtctaggc cccccgaacc 12301 acggggacgt ggttttcctt tgaaaaacac gataatacca tggtgagcaa gggcgaggag 12361 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg 12421 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag 12481 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc 12541 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac 12601 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc 12661 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg 12721 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc 12781 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag 12841 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag 12901 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc 12961 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc 13021 accggcggca tggacgagct gtacaagtag taaagcggcc gcgactctag agtcgagtct 13081 agatatcaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc 13141 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt 13201 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt 13261 atttggttta gagtttggca acatatgcca tatgctggct gccatgaaca aaggtggcta 13321 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa 13381 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca 13441 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc 13501 ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagctgatc 13561 tgcgaagata cggccacggg tgctcttgat cctgtggctg attttggact gtgctgctcg 13621 cagctgctga tgaatcacat acttcctcca ttttcttcca ctgattgact gttataattt 13681 ccctaatttc caggtcaagg tgctgtgcat tgtggtaata gatgtgacat gacgtcactt 13741 ccaaaggacc aatgaacatg tctgaccaat ttcatataat gtgaaaacga ttttcatagg 13801 cagaataaat aacatttaaa ttaaactggg catcagcgca attcaattgg tttggtaata 13861 gcaagggaaa atagaatgaa gtgatctcca aaaaataagt actttttgac tgtaaataaa 13921 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag 13981 tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca 14041 gtaatcaagt aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttcag 14101 caagcccagc agatccacta gttctagagc ggccgccacc gcggtggagc tccagctttt 14161 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg 14221 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 14281 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 14341 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 14401 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 14461 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 14521 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 14581 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 14641 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 14701 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 14761 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 14821 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 14881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 14941 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 15001 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 15061 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 15121 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 15181 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 15241 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 15301 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 15361 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 15421 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 15481 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 15541 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 15601 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 15661 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 15721 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 15781 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 15841 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 15901 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 15961 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 16021 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 16081 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 16141 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 16201 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 16261 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 16321 gggttccgcg cacatttccc cgaaaagtg //