LOCUS Exported 6525 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Active Wnt8B-V5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6525) 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 6525) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6525 /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 2015..2056 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" primer_bind complement(2096..2115) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 2140..2188 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 2390..2818 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2477..2496) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2687..2708 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2827..2847) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2832..3161 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3012..3147 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3074..3093 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3228..4022 /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(3282..3301) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3892..3911 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4198..4319 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4235..4254) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4289..4308 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4368..4384) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4368..4384) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4381..4403) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4392..4408 /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(4416..4446) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4461..4482 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4599..4616) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4770..5358) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4850..4869) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5529..6389) /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 6152..6171 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6390..6494) /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 tcaccatgtt 961 tctttcaaag ccttctgtgt acatctgtct tttcacctgt gtcctccaac tcagccacag 1021 ctggtcggtg aacaatttcc tgatgactgg tccaaaggct tacctgattt actccagcag 1081 tgtggcagct ggtgcccaga gtggtattga agaatgcaag tatcagtttg cctgggaccg 1141 ctggaactgc cctgagagag ccctgcagct gtccagccat ggtgggcttc gcagtgccaa 1201 tcgggagaca gcatttgtgc atgccatcag ttctgctgga gtcatgtaca ccctgactag 1261 aaactgcagc cttggagatt ttgataactg tggctgtgat gactcccgca acgggcaact 1321 ggggggacaa ggctggctgt ggggaggctg cagtgacaat gtgggcttcg gagaggcgat 1381 ttccaagcag tttgtcgatg ccctggaaac aggacaggat gcacgggcag ccatgaacct 1441 gcacaacaac gaggctggcc gcaaggcggt gaagggcacc atgaaacgca cgtgcaagtg 1501 ccacggcgtg tctggcagct gcaccacgca gacctgttgg ctgcagctgc ccgagttccg 1561 cgaggtgggc gcgcacctga aggagaagta ccacgcagca ctcaaggtgg acctgctgca 1621 gggtgctggc aacagcgcgg ccggccgcgg cgccatcgcc gacacctttc gctccatctc 1681 tacccgggag ctggtgcacc tggaggactc cccggactac tgcctggaga acaaaacgct 1741 agggctgctg ggcaccgaag gccgagagtg cctaaggcgc gggcgggccc tgggtcgctg 1801 ggaacgccgc agctgccgcc ggctctgcgg ggactgcggg ctggcggtgg aggagcgccg 1861 ggccgagacc gtgtccagct gcaactgcaa gttccactgg tgctgcgcag tccgctgcga 1921 gcagtgccgc cgtagggtca ccaagtactt ctgtagccgc gcagagcggc cgcggggggg 1981 cgctgcgcac aaacccggga gaaaacccgg cggaggtaag cctatcccta accctctcct 2041 cggtctcgat tctacgcgta ccggttagta atgagtttaa acgggggagg ctaactgaaa 2101 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata 2161 aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac 2221 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc 2281 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca 2341 ggccctgcca tagcagatct gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt 2401 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 2461 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 2521 tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag tgctttacgg 2581 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 2641 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 2701 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 2761 gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa 2821 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 2881 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 2941 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3001 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3061 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga 3121 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta 3181 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag 3241 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 3301 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 3361 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag 3421 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 3481 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 3541 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 3601 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 3661 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 3721 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg 3781 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 3841 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 3901 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 3961 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 4021 gagcgggact ctggggttcg cgaaatgacc gaccaagcga cgcccaacct gccatcacga 4081 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 4141 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac 4201 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 4261 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 4321 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt 4381 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4441 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 4501 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4561 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4621 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4681 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4741 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4801 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4861 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4921 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg 4981 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 5041 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 5101 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5161 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 5221 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5281 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5341 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5401 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5461 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5521 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5581 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5641 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5701 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5761 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5821 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5881 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5941 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6001 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6061 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6121 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6181 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6241 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6301 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 6361 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6421 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6481 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc //