LOCUS Exported 6582 bp ds-DNA circular SYN 14-APR-2022 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Active Wnt1-V5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6582) 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 6582) AUTHORS . TITLE Direct Submission JOURNAL Exported Apr 14, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6582 /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 2072..2113 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" primer_bind complement(2153..2172) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 2197..2245 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 2447..2875 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2534..2553) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2744..2765 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2884..2904) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2889..3218 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3069..3204 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3131..3150 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3285..4079 /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(3339..3358) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3949..3968 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4255..4376 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4292..4311) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4346..4365 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4425..4441) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4425..4441) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4438..4460) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4449..4465 /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(4473..4503) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4518..4539 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4656..4673) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4827..5415) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4907..4926) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5586..6446) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFATFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6209..6228 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6447..6551) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 acaactccga cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 901 taagctatca acaagtttgt acaaaaaagc aggctccgcg gccgccccct tcaccatggg 961 gctctgggcg ctgttgcctg gctgggtttc tgctacgctg ctgctggcgc tggccgctct 1021 gcccgcagcc ctggctgcca acagcagtgg ccgatggtgg ggtattgtga acgtagcctc 1081 ctccacgaac ctgcttacag actccaagag tctgcaactg gtactcgagc ccagtctgca 1141 gctgttgagc cgcaaacagc ggcgtctgat acgccaaaat ccggggatcc tgcacagcgt 1201 gagtgggggg ctgcagagtg ccgtgcgcga gtgcaagtgg cagttccgga atcgccgctg 1261 gaactgtccc actgctccag ggccccacct cttcggcaag atcgtcaacc gaggctgtcg 1321 agaaacggcg tttatcttcg ctatcacctc cgccggggtc acccattcgg tggcgcgctc 1381 ctgctcagaa ggttccatcg aatcctgcac gtgtgactac cggcggcgcg gccccggggg 1441 ccccgactgg cactgggggg gctgcagcga caacattgac ttcggccgcc tcttcggccg 1501 ggagttcgtg gactccgggg agaaggggcg ggacctgcgc ttcctcatga accttcacaa 1561 caacgaggca ggccgtacga ccgtattctc cgagatgcgc caggagtgca agtgccacgg 1621 gatgtccggc tcatgcacgg tgcgcacgtg ctggatgcgg ctgcccacgc tgcgcgccgt 1681 gggcgatgtg ctgcgcgacc gcttcgacgg cgcctcgcgc gtcctgtacg gcaaccgcgg 1741 cagcaaccgc gcttcgcggg cggagctgct gcgcctggag ccggaagacc cggcccacaa 1801 accgccctcc ccccacgacc tcgtctactt cgagaaatcg cccaacttct gcacgtacag 1861 cggacgcctg ggcacagcag gcacggcagg gcgcgcctgt aacagctcgt cgcccgcgct 1921 ggacggctgc gagctgctct gctgcggcag gggccaccgc acgcgcacgc agcgcgtcac 1981 cgagcgctgc aactgcacct tccactggtg ctgccacgtc agctgccgca actgcacgca 2041 cacgcgcgta ctgcacgagt gtctgggcgg aggtaagcct atccctaacc ctctcctcgg 2101 tctcgattct acgcgtaccg gttagtaatg agtttaaacg ggggaggcta actgaaacac 2161 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa 2221 cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct 2281 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca 2341 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc 2401 cctgccatag cagatctgcg cagctggggc tctagggggt atccccacgc gccctgtagc 2461 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 2521 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 2581 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 2641 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 2701 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 2761 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 2821 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc 2881 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta 2941 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 3001 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa 3061 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 3121 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt 3181 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat 3241 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 3301 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 3361 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 3421 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 3481 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 3541 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 3601 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 3661 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 3721 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 3781 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 3841 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 3901 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 3961 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 4021 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 4081 cgggactctg gggttcgcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 4141 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 4201 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 4261 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 4321 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 4381 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 4441 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4501 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4561 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4621 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4681 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4741 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4801 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 4861 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 4921 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 4981 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 5041 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 5101 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 5161 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 5221 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 5281 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5341 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5401 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5461 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5521 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5581 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 5641 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 5701 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 5761 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 5821 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 5881 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 5941 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 6001 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 6061 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 6121 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 6181 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6241 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 6301 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 6361 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg tcgcaaaaaa gggaataagg 6421 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 6481 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 6541 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc //