LOCUS Exported 5981 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of GST-tagged beta2 subunit of AP2 complex (hinge and ear) long splice form. ACCESSION . VERSION . KEYWORDS GST-beta2(616-951) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5981) 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 5981) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5981 /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(2031..2053) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2198..2216) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2284..2388 /gene="bla" /label=AmpR promoter CDS 2389..3249 /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(2607..2626) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3420..4008 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3909..3928 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4162..4179 /label=L4440 /note="L4440 vector, forward primer" promoter 4252..4329 /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 4330..5412 /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(4349..4368) /label=LacI-R /note="LacI, reverse primer" protein_bind 5425..5446 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5461..5491 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5499..5515 /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 5523..5539 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5523..5539 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 5535..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(5555..5572) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5555..5571) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5564..5586) /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 atcttctagg ggatctttta aaccttgacc 1021 tcggtccccc agtcaatgtg ccacaggtgt cctccatgca gatgggagca gtggatctcc 1081 taggaggagg actagatagt ctgcttggca gtgaccttgg cgggggcatt ggaggaagtc 1141 cggcagtggg acaatccttc atcccatcat cggtccctgc aacctttgct ccttcaccta 1201 cacctgctgt ggtcagcagt ggactgaatg acctgtttga actctccaca gggataggca 1261 tggcacctgg tggatatgtg gctcctaagg ctgtctggct acctgcagta aaggctaaag 1321 gcttggagat ttccggaaca tttactcacc gccaagggca catctatatg gaaatgaact 1381 tcaccaataa agctctgcag cacatgacag attttgcaat ccagtttaac aaaaatagct 1441 ttggtgtcat ccccagcact cctctggcca tccatacacc actgatgcca aaccagagca 1501 ttgatgtctc cctgcctctc aataccttgg gcccagtcat gaagatggaa cctctgaata 1561 acctccaggt ggctgtgaaa aacaatatcg atgtcttcta cttcagctgc ctcatcccac 1621 tcaatgtgct ttttgtagaa gatggcaaaa tggagcgcca ggtcttcctt gcaacatgga 1681 aggatattcc caatgaaaat gaacttcagt ttcagattaa ggaatgtcat ttaaatgctg 1741 acactgtttc cagcaagttg caaaacaaca atgtttatac tattgccaag aggaatgtgg 1801 aagggcagga catgctgtac caatccctga agctcactaa tggcatttgg attttggccg 1861 aactacgtat ccagccagga aaccccaatt acacgctgtc actgaagtgt agagctcctg 1921 aagtctctca atacatctat caggtctacg acagcatttt gaaaaactaa gcggccgcat 1981 cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2041 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2101 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt 2161 agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta 2221 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 2281 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 2341 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 2401 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 2461 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 2521 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 2581 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc 2641 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2701 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2761 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2821 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 2881 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg 2941 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 3001 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 3061 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 3121 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 3181 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 3241 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 3301 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 3361 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 3421 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 3481 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 3541 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 3601 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 3661 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3721 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3781 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 3841 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 3901 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 3961 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 4021 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 4081 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 4141 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 4201 cggtattttc tccttacgca tctgtgcggt atttcacacc gcataaattc cgacaccatc 4261 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg 4321 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat 4381 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa 4441 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg 4501 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg 4561 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg 4621 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa 4681 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa 4741 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac 4801 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg 4861 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt 4921 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa 4981 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc 5041 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt 5101 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa 5161 gacagctcat gttatatccc gccgtcaacc accatcaaac aggattttcg cctgctgggg 5221 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag 5281 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc 5341 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 5401 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc 5461 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 5521 cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg 5581 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 5641 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 5701 atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg 5761 agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt 5821 acgatgcgcc catctacacc aacgtaacct atcccattac ggtcaatccg ccgtttgttc 5881 ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac 5941 aggaaggcca gacgcgaatt atttttgatg gcgttggaat t //