LOCUS       sequence-403298-        6027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of VP1 protein from coxsackievirus A4 (CAV4). 
            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 6027)
  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 6027)
  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_CVA4_VP1
FEATURES             Location/Qualifiers
     source          1..6027
                     /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             1846..1863
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2075..2097)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2242..2260)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2328..2432
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2433..2501,2502..3293)
                     /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(2651..2670)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3464..4052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3953..3972
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4206..4223
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4296..4373
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4374..5456
                     /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(4393..4412)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5505..5510,5511..5528,5529..5535)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5543..5559
                     /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     5567..5583
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5567..5583
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5579..6027,1..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(5599..5616)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5599..5615)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5608..5630)
                     /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 tccgggttag gtcagatgct tgaaagcatg
      961 attgacaaca cagtccgtga aacggtgggg gcggcaacgt ctagagacgc tctcccaaac
     1021 actgaagcca gtggaccagc acactccaag gaaattccgg cactcaccgc agtggaaact
     1081 ggggccacaa atccactagt cccttctgat acagtgcaaa ccagacatgt tgtacaacat
     1141 aggtcaaggt cagagtctag catagagtct ttcttcgcgc ggggtgcatg cgtggccatt
     1201 ataaccgtgg ataactcagc ttccaccaag aataaggata agctatttac agtgtggaag
     1261 atcacttata aagatactgt ccagttacgg aggaaattgg agttcttcac ctattctaga
     1321 tttgatatgg aatttacctt tgtggttact gcaaatttca ctgagactaa caatgggcat
     1381 gccttaaatc aagtgtacca aattatgtac gtaccaccag gcgctccagt gcccgagaaa
     1441 tgggacgact acacatggca aacctcatca aatccatcaa tcttttacac ctacggaaca
     1501 gctccagccc ggatctcggt accgtatgtt ggtatttcga acgcctattc acacttttac
     1561 gacggttttt ccaaagtacc actgaaggac cagtcggcag cactaggtga ctccctctat
     1621 ggtgcagcat ctctaaatga cttcggtatt ttggctgtta gagtagtcaa tgatcacaac
     1681 ccgaccaagg tcacctccaa aatcagagtg tatctaaaac ccaaacacat cagagtctgg
     1741 tgcccgcgtc caccgagggc agtggcgtac tacggccctg gagtggatta caaggatggt
     1801 acgcttacac ccctctccac caaggatctg accacatatc ccgggcacca tcatcaccac
     1861 catcccgggt aataggaatt cacgcacgcg gccgccgagg gcagtggcgt actacggccc
     1921 tggagtggat tacaaggatg gtacgcttac acccctctcc accaaggatc tgaccacata
     1981 tcccgggcac cacatcacca ccatcccggg taataggaat tcatcgtgac tgactgacga
     2041 tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     2101 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     2161 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     2221 ataattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2281 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2341 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2401 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2461 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2521 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2581 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2641 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2701 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2761 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2821 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2881 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2941 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     3001 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3061 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3121 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3181 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3241 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3301 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3361 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3421 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3481 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3541 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3601 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3661 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3721 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3781 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3841 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3901 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3961 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4021 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4081 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4141 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4201 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4261 cgcatctgtg cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct
     4321 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     4381 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     4441 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     4501 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     4561 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     4621 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     4681 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     4741 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     4801 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     4861 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     4921 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     4981 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     5041 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     5101 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     5161 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     5221 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     5281 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     5341 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5401 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5461 aacgcaatta atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5521 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5581 gaccatgatt acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     5641 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     5701 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt
     5761 tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga
     5821 ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta
     5881 caccaacgtg acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac
     5941 gggttgttac tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg
     6001 aattattttt gatggcgttg gaattag
//
