LOCUS       sequence-193273-        5981 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 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 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)Y815A
FEATURES             Location/Qualifiers
     source          1..5981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(190..195,196..211,212..218)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    226..242
                     /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             265..918
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     876..898
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             925..948
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2038..2060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2205..2223)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2291..2395
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2396..2464,2465..3256)
                     /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(2614..2633)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3427..4015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3916..3935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4169..4186
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4259..4336
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4337..5419
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4356..4375)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5468..5473,5474..5491,5492..5498)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5506..5522
                     /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     5530..5546
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5530..5546
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5542..5981,1..88)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5562..5579)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5562..5578)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5571..5593)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
       61 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
      121 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
      181 aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac
      241 aatttcacac aggaaacagt attcatgtcc cctatactag gttattggaa aattaagggc
      301 cttgtgcaac ccactcgact tcttttggaa tatcttgaag aaaaatatga agagcatttg
      361 tatgagcgcg atgaaggtga taaatggcga aacaaaaagt ttgaattggg tttggagttt
      421 cccaatcttc cttattatat tgatggtgat gttaaattaa cacagtctat ggccatcata
      481 cgttatatag ctgacaagca caacatgttg ggtggttgtc caaaagagcg tgcagagatt
      541 tcaatgcttg aaggagcggt tttggatatt agatacggtg tttcgagaat tgcatatagt
      601 aaagactttg aaactctcaa agttgatttt cttagcaagc tacctgaaat gctgaaaatg
      661 ttcgaagatc gtttatgtca taaaacatat ttaaatggtg atcatgtaac ccatcctgac
      721 ttcatgttgt atgacgctct tgatgttgtt ttatacatgg acccaatgtg cctggatgcg
      781 ttcccaaaat tagtttgttt taaaaaacgt attgaagcta tcccacaaat tgataagtac
      841 ttgaaatcca gcaagtatat agcatggcct ttgcagggct ggcaagccac gtttggtggt
      901 ggcgaccatc ctccaaaatc ggatctggaa gttctgttcc aggggcccct gggatccccg
      961 gaattcgaac agcctcaggt tatcccctct caaggtgatc ttctagggga tcttttaaac
     1021 cttgacctcg gtcccccagt caatgtgcca caggtgtcct ccatgcagat gggagcagtg
     1081 gatctcctag gaggaggact agatagtctg cttggcagtg accttggcgg gggcattgga
     1141 ggaagtccgg cagtgggaca atccttcatc ccatcatcgg tccctgcaac ctttgctcct
     1201 tcacctacac ctgctgtggt cagcagtgga ctgaatgacc tgtttgaact ctccacaggg
     1261 ataggcatgg cacctggtgg atatgtggct cctaaggctg tctggctacc tgcagtaaag
     1321 gctaaaggct tggagatttc cggaacattt actcaccgcc aagggcacat ctatatggaa
     1381 atgaacttca ccaataaagc tctgcagcac atgacagatt ttgcaatcca gtttaacaaa
     1441 aatagctttg gtgtcatccc cagcactcct ctggccatcc atacaccact gatgccaaac
     1501 cagagcattg atgtctccct gcctctcaat accttgggcc cagtcatgaa gatggaacct
     1561 ctgaataacc tccaggtggc tgtgaaaaac aatatcgatg tcttcgcctt cagctgcctc
     1621 atcccactca atgtgctttt tgtagaagat ggcaaaatgg agcgccaggt cttccttgca
     1681 acatggaagg atattcccaa tgaaaatgaa cttcagtttc agattaagga atgtcattta
     1741 aatgctgaca ctgtttccag caagttgcaa aacaacaatg tttatactat tgccaagagg
     1801 aatgtggaag ggcaggacat gctgtaccaa tccctgaagc tcactaatgg catttggatt
     1861 ttggccgaac tacgtatcca gccaggaaac cccaattaca cgctgtcact gaagtgtaga
     1921 gctccagaag tctctcaata catctatcag gtctacgaca gcattttgaa aaactaagcg
     1981 gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     2041 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     2101 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     2161 gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct
     2221 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2281 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2341 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2401 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2461 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2521 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2581 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2641 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2701 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2761 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2821 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2881 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2941 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3001 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3061 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3121 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3181 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3241 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3301 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3361 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3421 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3481 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3541 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3601 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3661 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3721 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3781 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3841 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3901 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3961 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4021 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4081 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4141 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4201 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga
     4261 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4321 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4381 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4441 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4501 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4561 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     4621 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     4681 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     4741 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     4801 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     4861 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     4921 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     4981 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     5041 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     5101 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     5161 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5221 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5281 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc acgctagcgc ccaatacgca
     5341 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5401 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     5461 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     5521 aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa
     5581 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5641 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5701 agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc
     5761 tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg
     5821 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg
     5881 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc
     5941 tggctacagg aaggccagac gcgaattatt tttgatggcg t
//
