LOCUS       sequence-403316-        5856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of VP1 protein from echovirus 30 (E30). 
            Contains N-terminal GST-tag and C-terminal His6-tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5856)
  AUTHORS   Jouppila NVV, Lehtonen J, Seppala E, Puustinen L, Oikarinen S, 
            Laitinen OH, Knip M, Hyoty H, Hytonen VP
  TITLE     Assessment of Enterovirus Antibodies during Early Childhood Using a 
            Multiplex Immunoassay.
  JOURNAL   Microbiol Spectr. 2023 Jun 15;11(3):e0535222. doi: 
            10.1128/spectrum.05352-22. Epub 2023 May 25.
  PUBMED    37227147
REFERENCE   2  (bases 1 to 5856)
  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: "Microbiol 
            Spectr."; date: "2023-06-15"; volume: "11(3):e0535222. doi"; pages: 
            " 10.1128/spectrum.05352-22. Epub 2023 May 25"
COMMENT     Sequence Label: pGEX-2T_E30_VP1
FEATURES             Location/Qualifiers
     source          1..5856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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             256..909
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     867..889
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             916..933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1816..1833
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1904..1926)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2071..2089)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2157..2261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2262..2330,2331..3122)
                     /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(2480..2499)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3293..3881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3782..3801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4035..4052
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4125..4202
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4203..5285
                     /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(4222..4241)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5334..5339,5340..5357,5358..5364)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5372..5388
                     /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     5396..5412
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5396..5412
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5408..5856,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5428..5445)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5428..5444)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5437..5459)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      301 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      361 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      421 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      481 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      541 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      601 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      661 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      721 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      781 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      841 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      901 cctccaaaat cggatctggt tccgcgtgga tccaatgacc ctgaaagtgc actcaacagg
      961 gcagtgggca gggtggctga cactatagcc agtggacccg ttaacactga gcaaattcct
     1021 gctttaacag cagtggagac agggcacaca tcacaagtgg taccgagtga cacaatgcag
     1081 acacgacacg tggtcaacta ccacaccaga tcagagtcat caatagagaa ctttatgggt
     1141 agagcagcat gtgtgtacat cgctcagtac gccacagaga aggtcaacga tgagttagac
     1201 aggtacacca actgggaaat aacaaccaga caagtagcac agttgaggcg gaaactggaa
     1261 atgttcacat acatgaggtt tgaccttgag gtcacgtttg ttatcaccag ttcccagcgc
     1321 acttcaacca catacgcatc ggactcccct ccactaacgc accaagtgat gtatgtacca
     1381 ccaggaggcc caattcctag aagttatgaa gattttgcct ggcagacatc cactaaccca
     1441 agtgtgttct ggacagaagg caatgcccct cccaggatgt cgataccatt catgagtgtt
     1501 ggtaacgcat attgcaactt ttatgatgga tggtctcatt tcagccagag cggtgtgtac
     1561 ggatacacta ctttgaacaa catgggacac ctgtatttta gacatgtgaa caaatcaact
     1621 gcatatccag ttaacagtgt tgcccgcgtc tattttaagc ccaagcacgt gaaggcatgg
     1681 gtgcctcgtg cgccacgctt gtgcccatat ctgtatgcaa gaaatgtcaa ctttgatgtg
     1741 caaggggtaa ccgaatctcg gggcaaaatt acccttgacc gatcaactca caatcctttg
     1801 tcaaacactc ccgggcacca tcatcaccac catcccgggt aataggaatt catcgtgact
     1861 gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1921 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1981 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     2041 gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     2101 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     2161 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2221 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2281 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2341 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2401 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2461 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     2521 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2581 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2641 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2701 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2761 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2821 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2881 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2941 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     3001 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3061 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3121 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3181 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3241 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3301 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3361 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3421 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3481 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3541 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3601 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3661 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3721 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3781 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3841 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3901 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3961 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4021 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4081 ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc
     4141 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     4201 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     4261 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     4321 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     4381 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     4441 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     4501 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     4561 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     4621 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     4681 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     4741 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     4801 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     4861 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     4921 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     4981 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     5041 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     5101 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     5161 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     5221 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     5281 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac
     5341 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     5401 aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga
     5461 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5521 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5581 atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga
     5641 tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc
     5701 gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga
     5761 gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg
     5821 ccagacgcga attatttttg atggcgttgg aattag
//
