LOCUS       sequence-193274-        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(187..192,193..208,209..215)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    223..239
                     /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             262..915
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     873..895
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             922..945
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2008..2030)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2175..2193)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2261..2365
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2366..2434,2435..3226)
                     /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(2584..2603)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3397..3985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3886..3905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4139..4156
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4229..4306
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4307..5389
                     /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(4326..4345)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5438..5443,5444..5461,5462..5468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5476..5492
                     /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     5500..5516
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5500..5516
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5512..5954,1..85)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5532..5549)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5532..5548)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5541..5563)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 aattagctta tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc
       61 tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc
      121 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
      181 gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat
      241 ttcacacagg aaacagtatt catgtcccct atactaggtt attggaaaat taagggcctt
      301 gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat
      361 gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc
      421 aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt
      481 tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca
      541 atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa
      601 gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc
      661 gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc
      721 atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc
      781 ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg
      841 aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc
      901 gaccatcctc caaaatcgga tctggaagtt ctgttccagg ggcccctggg atccccggaa
      961 ttcgaacagc ctcaggttat cccctctcaa ggtgatctcg gtcccccagt caatgtgcca
     1021 caggtgtcct ccatgcagat gggagcagtg gatctcctag gaggaggact agatagtctg
     1081 cttggcagtg accttggcgg gggcattgga ggaagtccgg cagtgggaca atccttcatc
     1141 ccatcatcgg tccctgcaac ctttgctcct tcacctacac ctgctgtggt cagcagtgga
     1201 ctgaatgacc tgtttgaact ctccacaggg ataggcatgg cacctggtgg atatgtggct
     1261 cctaaggctg tctggctacc tgcagtaaag gctaaaggct tggagatttc cggaacattt
     1321 actcaccgcc aagggcacat ctatatggaa atgaacttca ccaataaagc tctgcagcac
     1381 atgacagatt ttgcaatcca gtttaacaaa aatagctttg gtgtcatccc cagcactcct
     1441 ctggccatcc atacaccact gatgccaaac cagagcattg atgtctccct gcctctcaat
     1501 accttgggcc cagtcatgaa gatggaacct ctgaataacc tccaggtggc tgtgaaaaac
     1561 aatatcgatg tcttctactt cagctgcctc atcccactca atgtgctttt tgtagaagat
     1621 ggcaaaatgg agcgccaggt cttccttgca acatggaagg atattcccaa tgaaaatgaa
     1681 cttcagtttc agattaagga atgtcattta aatgctgaca ctgtttccag caagttgcaa
     1741 aacaacaatg tttatactat tgccaagagg aatgtggaag ggcaggacat gctgtaccaa
     1801 tccctgaagc tcactaatgg catttggatt ttggccgaac tacgtatcca gccaggaaac
     1861 cccaattaca cgctgtcact gaagtgtaga gctcctgaag tctctcaata catctatcag
     1921 gtctacgaca gcattttgaa aaactaagcg gccgcatcgt gactgactga cgatctgcct
     1981 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2041 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2101 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     2161 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2221 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2281 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2341 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2401 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2461 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2521 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2581 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     2641 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2701 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2761 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2821 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2881 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2941 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3001 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3061 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3121 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3181 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3241 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3301 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3361 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3421 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3481 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3541 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3601 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3661 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3721 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3781 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3841 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3901 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3961 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4021 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4081 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4141 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4201 gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg
     4261 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     4321 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     4381 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     4441 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     4501 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     4561 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     4621 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4681 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4741 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     4801 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     4861 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     4921 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4981 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     5041 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     5101 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     5161 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     5221 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     5281 aagaaaaacc acgctagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     5341 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     5401 ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     5461 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     5521 attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5581 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5641 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg
     5701 tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat
     5761 actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac
     5821 gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt
     5881 tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt
     5941 tttgatggcg ttgg
//
