LOCUS Exported 13818 bp ds-DNA circular SYN 25-MAY-2021 DEFINITION Expresses a SYN-driven, intron-containing dCas9-KRAB-MeCP2 fusion in which the part of the dCas9 cassette is double floxed and in inverted orientation (DIO). Requires Cre-dependent recombination.. ACCESSION . VERSION . KEYWORDS lenti SYN-SVI-DIO-dCas9-KRAB-MeCP2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13818) TITLE A Cre-dependent CRISPR/dCas9 activation system for gene expression regulation in neurons JOURNAL Unpublished REFERENCE 2 (bases 1 to 13818) AUTHORS . TITLE Direct Submission JOURNAL Exported May 25, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13818 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 382..580 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 598..778 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 825..950 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1443..1676 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1861..1905 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 2203..2320 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2365..2812 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" protein_bind 2825..2858 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 2909..2942 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(4430..4450) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(4457..4522) /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" regulatory 4522..4531 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(4532..4565) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(4616..4649) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 4914..7412 /codon_start=1 /product="C-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(C) /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGD" CDS 7413..7460 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7521..7715 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEPWLV" CDS 7749..7769 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7779..8636 /codon_start=1 /product="transcriptional repression domain from rat methyl-CpG-binding protein 2 (Nan et al., 1997)" /label=MeCP2 /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV TERVS" misc_feature 8673..9261 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8726..8746) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9144..9155) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 9333..9566 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(9592..9609) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9598..9822 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9868..10296 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9955..9974) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10165..10186 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10305..10325) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10310..10639 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10490..10625 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10552..10571 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10687..10734 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10753..11127 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 11257..11378 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11294..11313) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11348..11367 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11427..11443) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11427..11443) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11440..11462) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11451..11467 /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(11475..11505) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11520..11541 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11658..11675) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11829..12417) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11909..11928) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12588..13448) /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 13211..13230 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13449..13553) /gene="bla" /label=AmpR promoter primer_bind complement(13628..13647) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2341 cagagatcca gtttggttaa ttaaagtgca agtgggtttt aggaccagga tgaggcgggg 2401 tgggggtgcc tacctgacga ccgaccccga cccactggac aagcacccaa cccccattcc 2461 ccaaattgcg catcccctat cagagagggg gaggggaaac aggatgcggc gaggcgcgtg 2521 cgcactgcca gcttcagcac cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca 2581 ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct 2641 tcccggccac cttggtcgcg tccgcgccgc cgccggccca gccggaccgc accacgcgag 2701 gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct 2761 gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc ctgagagcgc agaccggtgg 2821 taccataact tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta 2881 gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt 2941 atactaccca ggctagcccg gtgccaccat caccggatcc gggacctgaa ataaaagaca 3001 aaaagactaa acttacccag gcgaatctgc tgtttcccct ggccagaggg cccacgtagt 3061 aggggatgcg gaaggtcagg atcttctcga tcttttcccg gttgtccttc aggaatgggt 3121 aaaaatcttc ctgccgccgc agaatggcgt gcagctctcc caggtggatc tggtggggga 3181 tgctgccgtt gtcgaaggtc cgctgcttcc gcagcaggtc ctctctgttc agcttcacga 3241 gcagttcctc ggtgccgtcc atcttttcca ggatgggctt gatgaacttg tagaactctt 3301 cctggctggc tccgccgtca atgtagccgg cgtagccgtt cttgctctgg tcgaagaaaa 3361 tctctttgta cttctcaggc agctgctgcc gcacgagagc tttcagcagg gtcaggtcct 3421 ggtggtgctc gtcgtatctc ttgatcatag aggcgctcag gggggccttg gtgatctcgg 3481 tgttcactct caggatgtcg ctcagcagga tggcgtcgga caggttcttg gcggccagaa 3541 acaggtcggc gtactggtcg ccgatctggg ccagcaggtt gtccaggtcg tcgtcgtagg 3601 tgtccttgct cagctgcagt ttggcatcct cggccaggtc gaagttgctc ttgaagttgg 3661 gggtcaggcc caggctcagg gcaatcaggt tgccgaacag gccattcttc ttctcgccgg 3721 gcagctgggc gatcagattt tccagccgtc tgctcttgct cagtctggca gacaggatgg 3781 ccttggcgtc cacgccgctg gcgttgatgg ggttttcctc gaacagctgg ttgtaggtct 3841 gcaccagctg gatgaacagc ttgtccacgt cgctgttgtc ggggttcagg tcgccctcga 3901 tcaggaagtg gccccggaac ttgatcatgt gggccagggc cagatagatc agccgcaggt 3961 cggccttgtc ggtgctgtcc accagtttct ttctcaggtg gtagatggtg gggtacttct 4021 cgtggtaggc cacctcgtcc acgatgttgc cgaagatggg gtgccgctcg tgcttcttat 4081 cctcttccac caggaaggac tcttccagtc tgtggaagaa gctgtcgtcc accttggcca 4141 tctcgttgct gaagatctct tgcagatagc agatccggtt cttccgtctg gtgtatcttc 4201 ttctggcggt tctcttcagc cgggtggcct cggctgtttc gccgctgtcg aacagcaggg 4261 ctccgatcag gttcttcttg atgctgtgcc ggtcggtgtt gcccagcacc ttgaatttct 4321 tgctgggcac cttgtactcg tcggtgatca cggcccagcc cacagagttg gtgccgatgg 4381 ccaggccgat gctgtacttc ttgtcggctg ctgggactcc gtggataccg accttccgct 4441 tcttctttgg ggccatctta tcgtcatcgt ctttgtaatc aatatcatga tccttgtagt 4501 ctccgtcgtg gtccttatag tccatggtgg cataacttcg tataaagtat cctatacgaa 4561 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac 4621 ttcgtataat gtatgctata cgaagttatg tggtgcaaat caaagaactg ctcctcagtg 4681 gatgttgcct ttacttctag atgaccagaa agagcgagga aaccatcacc ccctggaact 4741 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 4801 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 4861 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 4921 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 4981 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 5041 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 5101 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 5161 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 5221 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 5281 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga 5341 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc 5401 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg 5461 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc 5521 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga 5581 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 5641 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 5701 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 5761 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 5821 tggacgctat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 5881 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 5941 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 6001 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 6061 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc 6121 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc 6181 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga 6241 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga 6301 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg 6361 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct 6421 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 6481 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 6541 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 6601 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 6661 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 6721 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 6781 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 6841 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 6901 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 6961 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 7021 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 7081 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 7141 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 7201 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 7261 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 7321 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 7381 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg 7441 gacaggccaa aaagaaaaag agtggtggag gaagtggcgg gtcagggtcg atggacgcga 7501 aatcacttac ggcatggtcg agaacactgg ttacgttcaa ggacgtgttt gtggacttta 7561 cacgtgagga gtggaaattg ctggatactg cgcaacaaat tgtgtatcga aatgtcatgc 7621 ttgagaatta caagaacctc gtcagtctcg gataccagtt gacgaaaccg gatgtgatcc 7681 ttaggctcga aaagggggaa gaaccttggc tggtatcggg aggtggttcg ggtggctctg 7741 gatcaagccc aaagaagaaa cggaaggtgg aagcctcagt gcaggtgaaa agggtgctgg 7801 aaaaatcccc cggcaaactc ctcgtgaaga tgcccttcca ggcttcccct ggcggaaaag 7861 gtgaaggggg tggcgcaacc acatctgccc aggtcatggt catcaagcga cctggaagga 7921 aaagaaaggc cgaggctgac cctcaggcca ttccaaagaa acggggacgc aagccagggt 7981 ccgtggtcgc agctgcagca gctgaggcta agaaaaaggc agtgaaggaa agctccatcc 8041 gcagtgtgca ggagactgtc ctgcccatca agaagaggaa gactagggag accgtgtcca 8101 tcgaggtcaa agaagtggtc aagcccctgc tcgtgtccac cctgggcgaa aaatctggaa 8161 aggggctcaa aacatgcaag tcacctggac ggaaaagcaa ggagtctagt ccaaaggggc 8221 gctcaagctc cgcttctagt ccccctaaaa aggaacacca tcaccatcac catcacgccg 8281 agtctcctaa ggctcctatg ccactgctcc caccacctcc accacctgag ccacagtcaa 8341 gcgaagaccc catcagccca cccgagcctc aggatctgtc ctctagtatt tgcaaagagg 8401 aaaagatgcc cagagcaggc agcctggaga gtgatggctg tccaaaagaa cccgccaaga 8461 cccagcctat ggtggcagcc gctgcaacta ccaccacaac cacaactacc acagtggccg 8521 aaaaatacaa gcatcgcggc gagggcgaac gaaaggacat tgtgtcaagc tccatgccca 8581 gacctaaccg ggaggaacca gtcgatagta ggacacccgt gactgagaga gtctcatagg 8641 aattcaattc taactagacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg 8701 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 8761 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 8821 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 8881 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 8941 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 9001 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 9061 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 9121 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 9181 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 9241 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga 9301 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag 9361 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga 9421 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct 9481 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt 9541 ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg 9601 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 9661 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 9721 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 9781 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 9841 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 9901 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 9961 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 10021 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 10081 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 10141 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 10201 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 10261 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 10321 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 10381 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 10441 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 10501 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 10561 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 10621 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 10681 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg 10741 aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc 10801 gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac 10861 gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag 10921 gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 10981 gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag 11041 atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg 11101 cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc 11161 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 11221 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 11281 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 11341 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 11401 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 11461 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 11521 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 11581 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 11641 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 11701 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 11761 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 11821 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 11881 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 11941 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 12001 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 12061 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 12121 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 12181 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 12241 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 12301 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 12361 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 12421 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 12481 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 12541 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 12601 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 12661 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 12721 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 12781 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 12841 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 12901 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 12961 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 13021 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 13081 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 13141 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 13201 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 13261 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 13321 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 13381 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 13441 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 13501 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 13561 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca 13621 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg 13681 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga 13741 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac 13801 gggccagata tacgcgtt //