LOCUS Exported 5954 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of GST-tagged beta2 subunit of AP2 complex (hinge and ear) long splice form, mutant. ACCESSION . VERSION . KEYWORDS GST-beta2(616-951)∆CBM SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5954) AUTHORS Wood LA, Larocque G, Clarke NI, Sarkar S, Royle SJ TITLE New tools for ""hot-wiring"" clathrin-mediated endocytosis with temporal and spatial precision. JOURNAL J Cell Biol. 2017 Sep 27. pii: jcb.201702188. doi: 10.1083/jcb.201702188. PUBMED 28954824 REFERENCE 2 (bases 1 to 5954) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5954 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 183..211 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 219..235 /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)." CDS 258..911 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 869..891 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 918..941 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" primer_bind complement(2004..2026) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2171..2189) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2257..2361 /gene="bla" /label=AmpR promoter CDS 2362..3222 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2580..2599) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3393..3981 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3882..3901 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4135..4152 /label=L4440 /note="L4440 vector, forward primer" promoter 4225..4302 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 4303..5385 /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(4322..4341) /label=LacI-R /note="LacI, reverse primer" protein_bind 5398..5419 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5434..5464 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5472..5488 /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 5496..5512 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5496..5512 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 5508..81 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR QPSEAVVWLCRS" primer_bind complement(5528..5545) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5528..5544) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5537..5559) /label=M13/pUC Forward /note="In lacZ gene" ORIGIN 1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg 61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt 121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc 181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca 241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc 301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc 361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc 421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata 481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc 541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact 601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag 661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt 721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa 781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat 841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc 901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ccggaattcg 961 aacagcctca ggttatcccc tctcaaggtg atctcggtcc cccagtcaat gtgccacagg 1021 tgtcctccat gcagatggga gcagtggatc tcctaggagg aggactagat agtctgcttg 1081 gcagtgacct tggcgggggc attggaggaa gtccggcagt gggacaatcc ttcatcccat 1141 catcggtccc tgcaaccttt gctccttcac ctacacctgc tgtggtcagc agtggactga 1201 atgacctgtt tgaactctcc acagggatag gcatggcacc tggtggatat gtggctccta 1261 aggctgtctg gctacctgca gtaaaggcta aaggcttgga gatttccgga acatttactc 1321 accgccaagg gcacatctat atggaaatga acttcaccaa taaagctctg cagcacatga 1381 cagattttgc aatccagttt aacaaaaata gctttggtgt catccccagc actcctctgg 1441 ccatccatac accactgatg ccaaaccaga gcattgatgt ctccctgcct ctcaatacct 1501 tgggcccagt catgaagatg gaacctctga ataacctcca ggtggctgtg aaaaacaata 1561 tcgatgtctt ctacttcagc tgcctcatcc cactcaatgt gctttttgta gaagatggca 1621 aaatggagcg ccaggtcttc cttgcaacat ggaaggatat tcccaatgaa aatgaacttc 1681 agtttcagat taaggaatgt catttaaatg ctgacactgt ttccagcaag ttgcaaaaca 1741 acaatgttta tactattgcc aagaggaatg tggaagggca ggacatgctg taccaatccc 1801 tgaagctcac taatggcatt tggattttgg ccgaactacg tatccagcca ggaaacccca 1861 attacacgct gtcactgaag tgtagagctc ctgaagtctc tcaatacatc tatcaggtct 1921 acgacagcat tttgaaaaac taagcggccg catcgtgact gactgacgat ctgcctcgcg 1981 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 2041 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc 2101 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta taattcttga 2161 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 2221 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 2281 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 2341 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 2401 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 2461 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 2521 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 2581 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata 2641 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 2701 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 2761 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 2821 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 2881 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact 2941 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 3001 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 3061 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 3121 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 3181 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 3241 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 3301 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 3361 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 3421 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 3481 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 3541 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 3601 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 3661 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 3721 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 3781 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 3841 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 3901 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 3961 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 4021 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 4081 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 4141 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc 4201 ggtatttcac accgcataaa ttccgacacc atcgaatggt gcaaaacctt tcgcggtatg 4261 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta 4321 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag 4381 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 4441 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt 4501 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc 4561 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc 4621 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat 4681 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta 4741 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt 4801 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg 4861 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact 4921 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt 4981 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac 5041 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg 5101 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtca 5161 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5221 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 5281 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 5341 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 5401 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 5461 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 5521 cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 5581 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 5641 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc 5701 cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag gccgatactg 5761 tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac accaacgtaa 5821 cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg ggttgttact 5881 cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga attatttttg 5941 atggcgttgg aatt //