LOCUS Exported 15470 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Triple pathway reporter, 3P-Luc; wnt-NLuc1.1, hedgehog-FLuc, notch-GLuc; plus CMV-eGFP. Gateway expression vector for lentivirus generation.. ACCESSION . VERSION . KEYWORDS pMuLE_EXPR_CMV-eGFP_TOP-NLuc1.1_12GLI-FLuc_CBF-GLuc SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15470) AUTHORS Maier J, Elmenofi S, Taschauer A, Anton M, Sami H, Ogris M TITLE Luminescent and fluorescent triple reporter plasmid constructs for Wnt, Hedgehog and Notch pathway. JOURNAL PLoS One. 2019 Dec 20;14(12):e0226570. doi: 10.1371/journal.pone.0226570. eCollection 2019. PUBMED 31860685 REFERENCE 2 (bases 1 to 15470) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15470 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 94..327 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 399..520 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" rep_origin 560..695 /label=SV40 ori /note="SV40 origin of replication" promoter complement(716..734) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(744..761) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(744..760) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(753..775) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 902..1357 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(989..1008) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1199..1220 /label=F1ori-F /note="F1 origin, forward primer" promoter 1383..1487 /gene="bla" /label=AmpR promoter CDS 1488..2348 /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 complement(1706..1725) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2519..3107 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3008..3027 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3261..3278 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3395..3416 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3431..3461 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3469..3485 /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)." primer_bind 3474..3496 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3493..3509 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3493..3509 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 3528..3548 /label=T3 /note="T3 promoter, forward primer" promoter 3530..3548 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 3576..3802 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 3803..3983 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 4030..4155 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 4648..4881 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 5066..5110 /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 5377..5493 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 5550..5574 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" enhancer 5602..5981 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5982..6185 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6136..6156 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" promoter 6230..6248 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 6284..6293 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6290..7009 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 6290..7006 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(6335..6356) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6596..6615) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6943..6964 /label=EGFP-C /note="EGFP, forward primer" protein_bind 7050..7070 /gene="mutant version of attB" /label=attB5 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" promoter 7215..7246 /label=minP /note="minimal TATA-box promoter with low basal activity" regulatory 7302..7311 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 7308..7823 /codon_start=1 /product="NanoLuc(R) luciferase" /label=Nluc /note="human codon-optimized" /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILA" polyA_signal 7872..7993 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind complement(8183..8203) /gene="mutant version of attB" /label=attB4 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" primer_bind 8356..8378 /label=pGEX 3' /note="pGEX vectors, reverse primer" polyA_signal 8509..8643 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 8737..8871 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 9112..9174 /label=Mini-TK promoter /note="minimal herpes simplex virus (HSV) thymidine kinase promoter" CDS 9230..10882 /codon_start=1 /gene="luc" /product="firefly luciferase" /label=luciferase /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL" primer_bind complement(9296..9314) /label=LucNrev /note="Firefly luciferase, reverse primer" intron 11013..11078 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" CDS 11208..11228 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 11653..11787 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind 12339..12359 /gene="mutant version of attB" /label=attB3 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" promoter 12599..12795 /label=SV40 promoter /note="SV40 early promoter" rep_origin 12646..12781 /label=SV40 ori /note="SV40 origin of replication" regulatory 12858..12867 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 12864..13421 /codon_start=1 /product="secreted Gaussia luciferase" /label=hGLuc /note="human codon-optimized" /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ RCATFASKIQGQVDKIKGAGGD" protein_bind complement(13450..13474) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 13505..14093 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(13558..13578) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(13976..13987) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" promoter 14107..14606 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(14142..14165) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 14539..14558 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 14648..15442 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(14702..14721) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 15312..15331 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 cgcgtatgca tctcgagggc ccggtacctt taagaccaat gacttacaag gcagctgtag 61 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctagctcac tcccaacgaa 121 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg 181 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 241 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 301 tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt 361 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata 421 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 481 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat 541 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 601 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct 661 attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat 721 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac 781 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 841 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 901 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 961 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 1021 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 1081 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 1141 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 1201 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 1261 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 1321 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg 1381 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 1441 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 1501 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 1561 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 1621 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 1681 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 1741 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 1801 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 1861 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 1921 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 1981 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 2041 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 2101 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 2161 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 2221 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 2281 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 2341 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 2401 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 2461 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 2521 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 2581 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 2641 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 2701 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 2761 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 2821 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 2881 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 2941 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 3001 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 3061 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 3121 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 3181 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 3241 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 3301 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 3361 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 3421 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 3481 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc 3541 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta 3601 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg 3661 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac 3721 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa 3781 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct 3841 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 3901 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 3961 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa 4021 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag 4081 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat 4141 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg 4201 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag 4261 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata 4321 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat 4381 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct 4441 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct 4501 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata 4561 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt 4621 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc 4681 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc 4741 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt 4801 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata 4861 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac 4921 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac 4981 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat 5041 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg 5101 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat 5161 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa 5221 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg 5281 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat 5341 tcgattagtg aacggatctc gacggtatcg gttaactttt aaaagaaaag gggggattgg 5401 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga 5461 attacaaaaa caaattacaa aattcaaaat tttatcgtcc agtgtggtgg aattgagtat 5521 tccagtgtgg tggaattctg cagatatcaa caagtttgta caaaaaagca ggctgcgatg 5581 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta 5641 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 5701 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 5761 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 5821 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 5881 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 5941 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 6001 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 6061 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 6121 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 6181 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc 6241 actataggga gacccaagct tggtaccgag ctcggatcct aaagccacca tggtgagcaa 6301 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 6361 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 6421 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 6481 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 6541 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 6601 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 6661 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 6721 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 6781 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 6841 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 6901 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 6961 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga 7021 ctctagagct cgagcatgca tctagaggac aactttgtat acaaaagttg ggagacccaa 7081 gcttggtacc gagctcttac gcgagatcaa agggggtaag atcaaagggg gtaagatcaa 7141 aggggcgcga gatcaaaggg ggtaagatca aaggggcgcg cccgcgtgct agcccgggct 7201 cgagatctag actctagagg gtatataatg gaagctcgaa ttccagcttg gcattccggt 7261 actgttggta aaaagcttgg caatccggta ctgttggtaa agccaccatg gtcttcacac 7321 tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg 7381 aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa 7441 ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg 7501 aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg 7561 tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta 7621 cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca 7681 aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga 7741 tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc 7801 tgtgcgaacg cattctggcg taaggccgcg actctagagt cggggcggcc ggccgcttcg 7861 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 7921 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 7981 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 8041 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc 8101 actagtaacg gccgccagtg tgctggaatt ctgcagatat ccatcacact ggcggccgct 8161 cgagcatgca tctagaggca cccaactttt ctatacaaag ttgggagacc caagcttggt 8221 accgagctct atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc 8281 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 8341 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 8401 gcgcgaggca ggatcagcca tatcacattt gtagaggttt tacttgcttt aaaaaacctc 8461 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttaa cttgtttatt 8521 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 8581 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg 8641 atcagccata tcacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 8701 ctgaacctga aacataaaat gaatgcaatt gttgttaact tgtttattgc agcttataat 8761 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 8821 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccaagctcag 8881 atccaagctt atcgataccg tcgagtgggt ggtctgggtg gtctgggtgg tctgggtggt 8941 cgagtgggtg gtctgggtgg tctgggtggt ctgggtggtc gagtgggtgg tctgggtggt 9001 ctgggtggtc tgggtggtcg acctcgagat ccggcaaacc ccgcccagcg tcttgtcatt 9061 ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt ccgaggtcca cttcgcatat 9121 taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc agcgacccgc ttaacagcgt 9181 caacagcgtg ccgcagatct gagcttggca ttccggtact gttggtaaaa tggaagacgc 9241 caaaaacata aagaaaggcc cggcgccatt ctatcctcta gaggatggaa ccgctggaga 9301 gcaactgcat aaggctatga agagatacgc cctggttcct ggaacaattg cttttacaga 9361 tgcacatatc gaggtgaaca tcacgtacgc ggaatacttc gaaatgtccg ttcggttggc 9421 agaagctatg aaacgatatg ggctgaatac aaatcacaga atcgtcgtat gcagtgaaaa 9481 ctctcttcaa ttctttatgc cggtgttggg cgcgttattt atcggagttg cagttgcgcc 9541 cgcgaacgac atttataatg aacgtgaatt gctcaacagt atgaacattt cgcagcctac 9601 cgtagtgttt gtttccaaaa aggggttgca aaaaattttg aacgtgcaaa aaaaattacc 9661 aataatccag aaaattatta tcatggattc taaaacggat taccagggat ttcagtcgat 9721 gtacacgttc gtcacatctc atctacctcc cggttttaat gaatacgatt ttgtaccaga 9781 gtcctttgat cgtgacaaaa caattgcact gataatgaat tcctctggat ctactgggtt 9841 acctaagggt gtggcccttc cgcatagaac tgcctgcgtc agattctcgc atgccagaga 9901 tcctattttt ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca 9961 tcacggtttt ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt 10021 aatgtataga tttgaagaag agctgttttt acgatccctt caggattaca aaattcaaag 10081 tgcgttgcta gtaccaaccc tattttcatt cttcgccaaa agcactctga ttgacaaata 10141 cgatttatct aatttacacg aaattgcttc tgggggcgca cctctttcga aagaagtcgg 10201 ggaagcggtt gcaaaacgct tccatcttcc agggatacga caaggatatg ggctcactga 10261 gactacatca gctattctga ttacacccga gggggatgat aaaccgggcg cggtcggtaa 10321 agttgttcca ttttttgaag cgaaggttgt ggatctggat accgggaaaa cgctgggcgt 10381 taatcagaga ggcgaattat gtgtcagagg acctatgatt atgtccggtt atgtaaacaa 10441 tccggaagcg accaacgcct tgattgacaa ggatggatgg ctacattctg gagacatagc 10501 ttactgggac gaagacgaac acttcttcat agttgaccgc ttgaagtctt taattaaata 10561 caaaggatat caggtggccc ccgctgaatt ggaatcgata ttgttacaac accccaacat 10621 cttcgacgcg ggcgtggcag gtcttcccga cgatgacgcc ggtgaacttc ccgccgccgt 10681 tgttgttttg gagcacggaa agacgatgac ggaaaaagag atcgtggatt acgtcgccag 10741 tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg tttgtggacg aagtaccgaa 10801 aggtcttacc ggaaaactcg acgcaagaaa aatcagagag atcctcataa aggccaagaa 10861 gggcggaaag tccaaattgt aaaatgtaac tgtattcagc gatgacgaaa ttcttagcta 10921 ttgtaatact ctagaggatc tttgtgaagg aaccttactt ctgtggtgtg acataattgg 10981 acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta agtgtataat 11041 gtgttaaact actgattcta attgtttgtg tattttagat tccaacctat ggaactgatg 11101 aatgggagca gtggtggaat gcctttaatg aggaaaacct gttttgctca gaagaaatgc 11161 catctagtga tgatgaggct actgctgact ctcaacattc tactcctcca aaaaagaaga 11221 gaaaggtaga agaccccaag gactttcctt cagaattgct aagttttttg agtcatgctg 11281 tgtttagtaa tagaactctt gcttgctttg ctatttacac cacaaaggaa aaagctgcac 11341 tgctatacaa gaaaattatg gaaaaatatt ctgtaacctt tataagtagg cataacagtt 11401 ataatcataa catactgttt tttcttactc cacacaggca tagagtgtct gctattaata 11461 actatgctca aaaattgtgt acctttagct ttttaatttg taaaggggtt aataaggaat 11521 atttgatgta tagtgccttg actagagatc ataatcagcc ataccacatt tgtagaggtt 11581 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca 11641 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 11701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 11761 atcaatgtat cttatcatgt ctggatcgat ccccaggaag ctcctctgtg tcctcataaa 11821 ccctaacctc ctctacttga gaggacattc caatcatagg ctgcccatcc accctctgtg 11881 tcctcctgtt aattaggtca cttaacaaaa aggaaattgg gtaggggttt ttcacagacc 11941 gctttctaag ggtaatttta aaatatctgg gaagtccctt ccactgctgt gttccagaag 12001 tgttggtaaa cagcccacaa atgtcaacag cagaaacata caagctgtca gctttgcaca 12061 agggcccaac accctgctca tcaagaagca ctgtggttgc tgtgttagta atgtgcaaaa 12121 caggaggcac attttcccca cctgtgtagg ttccaaaata tctagtgttt tcatttttac 12181 ttggatcagg aacccagcac tccactggat aagcattatc cttatccaaa acagccttgt 12241 ggtcagtgtt catctgctga ctagtaacgg ccgccagtgt gctggaattc tgcagatatc 12301 catcacactg gcggccgctc gagcatgcat ctagaggaca actttgtata ataaagttgg 12361 gagacccaag cttggtaccg agctcggatc cactagtaac ggccgccagt gtgctggaat 12421 tcgtattacc gccatgcatt agttattaat cgagatctgg tgtaaacacg ccgtgggaaa 12481 aaatttatgg atctggagta acacgccgtg ggaaaaaatt tatggatctg gagtaacacg 12541 ccgtgggaaa aaatttatgg atctggagta acacgccgtg ggaaaaaatt tatggatctg 12601 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 12661 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 12721 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 12781 ctaggctttt gcaaaaagct ggctagcgct accggactca gatctcgagc tcaagcttgg 12841 taccgagctc ggatccagcc accatgggag tcaaagttct gtttgccctg atctgcatcg 12901 ctgtggccga ggccaagccc accgagaaca acgaagactt caacatcgtg gccgtggcca 12961 gcaacttcgc gaccacggat ctcgatgctg accgcgggaa gttgcccggc aagaagctgc 13021 cgctggaggt gctcaaagag atggaagcca atgcccggaa agctggctgc accaggggct 13081 gtctgatctg cctgtcccac atcaagtgca cgcccaagat gaagaagttc atcccaggac 13141 gctgccacac ctacgaaggc gacaaagagt ccgcacaggg cggcataggc gaggcgatcg 13201 tcgacattcc tgagattcct gggttcaagg acttggagcc catggagcag ttcatcgcac 13261 aggtcgatct gtgtgtggac tgcacaactg gctgcctcaa agggcttgcc aacgtgcagt 13321 gttctgacct gctcaagaag tggctgccgc aacgctgtgc gacctttgcc agcaagatcc 13381 agggccaggt ggacaagatc aagggggccg gtggtgacta agcggccgct cgagcatgca 13441 tctagaggta cccagctttc ttgtacaaag tggttgatat ccagcacagt ggcggccgct 13501 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 13561 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 13621 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 13681 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 13741 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 13801 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 13861 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct 13921 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 13981 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 14041 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat 14101 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc 14161 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg 14221 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc 14281 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag 14341 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag 14401 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg 14461 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa 14521 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct 14581 cctcttcctc atctccgggc ctttcgacct gcagcccaag cttaccacac tcctgcaggc 14641 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 14701 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 14761 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 14821 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 14881 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 14941 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 15001 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 15061 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 15121 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 15181 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 15241 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 15301 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 15361 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 15421 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga //