LOCUS       sequence-189390-        5954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of GST-tagged beta2 subunit of AP2 complex 
            (hinge and ear) long splice form, mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol. 2017 Sep 27. pii: jcb.201702188. doi: 10.1083/jcb.201702188."
COMMENT     Sequence Label: GST-beta2(616-951)∆CBM
FEATURES             Location/Qualifiers
     source          1..5954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..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             join(2362..2430,2431..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        join(5434..5439,5440..5457,5458..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             join(5508..5954,1..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
//
