LOCUS       sequence-189389-        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(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(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             join(2389..2457,2458..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        join(5461..5466,5467..5484,5485..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             join(5535..5981,1..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(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 atgtcttcgc 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
//
