LOCUS       sequence-192860-        5044 bp    DNA     circular SYN 15-APR-2025
DEFINITION  GST-STp expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5044)
  AUTHORS   Zhu Y, Luo Q, Davis SM, Westra C, Vickers TJ, Fleckenstein JM
  TITLE     Molecular determinants of enterotoxigenic Escherichia coli 
            heat-stable toxin secretion and delivery.
  JOURNAL   Infect Immun. 2018 Aug 20. pii: IAI.00526-18. doi: 
            10.1128/IAI.00526-18.
  PUBMED    30126899
REFERENCE   2  (bases 1 to 5044)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Infect 
            Immun. 2018 Aug 20. pii: IAI.00526-18. doi: 10.1128/IAI.00526-18."
COMMENT     Sequence Label: pCW003
FEATURES             Location/Qualifiers
     source          1..5044
                     /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..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1094..1116)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1261..1279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1347..1451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1452..1520,1521..2312)
                     /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(1670..1689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2483..3071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2972..2991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3225..3242
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3315..3392
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3393..4475
                     /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(3412..3431)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4524..4529,4530..4547,4548..4554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4562..4578
                     /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     4586..4602
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4586..4602
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4598..5044,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4618..4635)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4618..4634)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4627..4649)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga 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 gttccgcgtg gatccgatcc ccgggtaccg agctcgaaca
      961 cattttactg ctgtgaactt tgttgtaatc ctgcctgtgc tggatgttat tgagaattcc
     1021 cgggtcgact cgagcggccg catcgtgact gactgacgat ctgcctcgcg cgtttcggtg
     1081 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     1141 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     1201 gcgcagccat gacccagtca cgtagcgata gcggagtgta taattcttga agacgaaagg
     1261 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     1321 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1381 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1441 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1501 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1561 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1621 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1681 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1741 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1801 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1861 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1921 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1981 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2041 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2101 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2161 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2221 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2281 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2341 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2401 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2461 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2521 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2581 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2641 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2701 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2761 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2821 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2881 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2941 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3001 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3061 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3121 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3181 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3241 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3301 accgcataaa ttccgacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc
     3361 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     3421 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     3481 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     3541 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     3601 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     3661 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     3721 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     3781 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     3841 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     3901 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     3961 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     4021 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     4081 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     4141 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg
     4201 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca
     4261 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     4321 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     4381 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     4441 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag
     4501 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     4561 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cggattcact
     4621 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4681 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4741 ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc cggcaccaga
     4801 agcggtgccg gaaagctggc tggagtgcga tcttcctgag gccgatactg tcgtcgtccc
     4861 ctcaaactgg cagatgcacg gttacgatgc gcccatctac accaacgtga cctatcccat
     4921 tacggtcaat ccgccgtttg ttcccacgga gaatccgacg ggttgttact cgctcacatt
     4981 taatgttgat gaaagctggc tacaggaagg ccagacgcga attatttttg atggcgttgg
     5041 aatt
//
