LOCUS       sequence-193269-        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.
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)
FEATURES             Location/Qualifiers
     source          1..5981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(184..189,190..205,206..212)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    220..236
                     /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             259..912
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     870..892
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             919..942
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2032..2054)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2199..2217)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2285..2389
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2390..2458,2459..3250)
                     /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(2608..2627)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3421..4009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3910..3929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4163..4180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4253..4330
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4331..5413
                     /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(4350..4369)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5462..5467,5468..5485,5486..5492)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5500..5516
                     /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     5524..5540
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5524..5540
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5536..5981,1..82)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5556..5573)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5556..5572)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5565..5587)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tagcttatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
       61 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
      121 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
      181 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
      241 acacaggaaa cagtattcat gtcccctata ctaggttatt ggaaaattaa gggccttgtg
      301 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
      361 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
      421 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
      481 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
      541 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
      601 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
      661 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
      721 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
      781 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
      841 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
      901 catcctccaa aatcggatct ggaagttctg ttccaggggc ccctgggatc cccggaattc
      961 gaacagcctc aggttatccc ctctcaaggt gatcttctag gggatctttt aaaccttgac
     1021 ctcggtcccc cagtcaatgt gccacaggtg tcctccatgc agatgggagc agtggatctc
     1081 ctaggaggag gactagatag tctgcttggc agtgaccttg gcgggggcat tggaggaagt
     1141 ccggcagtgg gacaatcctt catcccatca tcggtccctg caacctttgc tccttcacct
     1201 acacctgctg tggtcagcag tggactgaat gacctgtttg aactctccac agggataggc
     1261 atggcacctg gtggatatgt ggctcctaag gctgtctggc tacctgcagt aaaggctaaa
     1321 ggcttggaga tttccggaac atttactcac cgccaagggc acatctatat ggaaatgaac
     1381 ttcaccaata aagctctgca gcacatgaca gattttgcaa tccagtttaa caaaaatagc
     1441 tttggtgtca tccccagcac tcctctggcc atccatacac cactgatgcc aaaccagagc
     1501 attgatgtct ccctgcctct caataccttg ggcccagtca tgaagatgga acctctgaat
     1561 aacctccagg tggctgtgaa aaacaatatc gatgtcttct acttcagctg cctcatccca
     1621 ctcaatgtgc tttttgtaga agatggcaaa atggagcgcc aggtcttcct tgcaacatgg
     1681 aaggatattc ccaatgaaaa tgaacttcag tttcagatta aggaatgtca tttaaatgct
     1741 gacactgttt ccagcaagtt gcaaaacaac aatgtttata ctattgccaa gaggaatgtg
     1801 gaagggcagg acatgctgta ccaatccctg aagctcacta atggcatttg gattttggcc
     1861 gaactacgta tccagccagg aaaccccaat tacacgctgt cactgaagtg tagagctcct
     1921 gaagtctctc aatacatcta tcaggtctac gacagcattt tgaaaaacta agcggccgca
     1981 tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2041 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2101 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     2161 tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt
     2221 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2281 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2341 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2401 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2461 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2521 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2581 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     2641 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2701 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2761 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2821 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2881 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2941 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3001 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3061 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3121 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3181 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3241 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3301 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3361 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3421 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3481 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3541 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3601 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3661 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3721 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3781 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3841 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3901 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3961 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4021 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4081 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4141 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4201 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat
     4261 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     4321 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     4381 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     4441 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     4501 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     4561 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     4621 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     4681 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     4741 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     4801 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     4861 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     4921 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     4981 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     5041 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     5101 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     5161 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     5221 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     5281 gctgttgccc gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc
     5341 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5401 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg
     5461 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5521 acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt
     5581 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     5641 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5701 aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg
     5761 gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt
     5821 tacgatgcgc ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt
     5881 cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta
     5941 caggaaggcc agacgcgaat tatttttgat ggcgttggaa t
//
