LOCUS Exported 6519 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Active Wnt7A-V5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6519) AUTHORS MacDonald BT, Hien A, Zhang X, Iranloye O, Virshup DM, Waterman ML, He X TITLE Disulfide Bond Requirements for Active Wnt Ligands. JOURNAL J Biol Chem. 2014 May 19. pii: jbc.M114.575027. PUBMED 24841207 REFERENCE 2 (bases 1 to 6519) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6519 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 911..935 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2009..2050 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" primer_bind complement(2090..2109) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 2134..2182 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 2384..2812 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2471..2490) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2681..2702 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2821..2841) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2826..3155 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3006..3141 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3068..3087 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3222..4016 /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(3276..3295) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3886..3905 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4192..4313 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4229..4248) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4283..4302 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4362..4378) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4362..4378) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4375..4397) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4386..4402 /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(4410..4440) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4455..4476 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4593..4610) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4764..5352) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4844..4863) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5523..6383) /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 6146..6165 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6384..6488) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 901 taagctatca acaagtttgt acaaaaaagc aggctccgcg gccgccccct tcaccatgaa 961 ccggaaagcg cggcgctgcc tgggccacct ctttctcagc ctgggcatgg tctacctccg 1021 gatcggtggc ttctcctcag tggtagctct gggcgcaagc atcatctgta acaagatccc 1081 aggcctggct cccagacagc gggcgatctg ccagagccgg cccgacgcca tcatcgtcat 1141 aggagaaggc tcacaaatgg gcctggacga gtgtcagttt cagttccgca atggccgctg 1201 gaactgctct gcactgggag agcgcaccgt cttcgggaag gagctcaaag tggggagccg 1261 ggaggctgcg ttcacctacg ccatcattgc cgccggcgtg gcccacgcca tcacagctgc 1321 ctgtacccag ggcaacctga gcgactgtgg ctgcgacaaa gagaagcaag gccagtacca 1381 ccgggacgag ggctggaagt ggggtggctg ctctgccgac atccgctacg gcatcggctt 1441 cgccaaggtc tttgtggatg cccgggagat caagcagaat gcccggactc tcatgaactt 1501 gcacaacaac gaggcaggcc gaaagatcct ggaggagaac atgaagctgg aatgtaagtg 1561 ccacggcgtg tcaggctcgt gcaccaccaa gacgtgctgg accacactgc cacagtttcg 1621 ggagctgggc tacgtgctca aggacaagta caacgaggcc gttcacgtgg agcctgtgcg 1681 tgccagccgc aacaagcggc ccaccttcct gaagatcaag aagccactgt cgtaccgcaa 1741 gcccatggac acggacctgg tgtacatcga gaagtcgccc aactactgcg aggaggaccc 1801 ggtgaccggc agtgtgggca cccagggccg cgcctgcaac aagacggctc cccaggccag 1861 cggctgtgac ctcatgtgct gtgggcgtgg ctacaacacc caccagtacg cccgcgtgtg 1921 gcagtgcaac tgtaagttcc actggtgctg ctatgtcaag tgcaacacgt gcagcgagcg 1981 cacggagatg tacacgtgca agggcggagg taagcctatc cctaaccctc tcctcggtct 2041 cgattctacg cgtaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga 2101 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc 2161 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc 2221 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc 2281 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct 2341 gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc 2401 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 2461 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 2521 cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc 2581 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 2641 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 2701 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt 2761 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt 2821 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2881 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 2941 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 3001 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 3061 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 3121 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca 3181 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat 3241 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 3301 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 3361 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc 3421 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 3481 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 3541 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 3601 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 3661 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 3721 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga 3781 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 3841 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 3901 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 3961 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg 4021 gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 4081 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 4141 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 4201 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4261 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 4321 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 4381 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 4441 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 4501 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 4561 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4621 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4681 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4741 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4801 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4861 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4921 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 4981 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 5041 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5101 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 5161 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 5221 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 5281 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 5341 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 5401 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 5461 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 5521 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 5581 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 5641 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 5701 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 5761 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 5821 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 5881 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 5941 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 6001 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 6061 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 6121 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 6181 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 6241 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 6301 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 6361 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 6421 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 6481 gttccgcgca catttccccg aaaagtgcca cctgacgtc //