LOCUS       sequence-403360-        5907 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression of VP1 protein from enterovirus D68 (EV-D68). 
            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 5907)
  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 5907)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_EV-D68_VP1
FEATURES             Location/Qualifiers
     source          1..5907
                     /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             1867..1884
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1955..1977)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2122..2140)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2208..2312
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2313..2381,2382..3173)
                     /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(2531..2550)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3344..3932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3833..3852
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4086..4103
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4176..4253
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4254..5336
                     /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(4273..4292)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5385..5390,5391..5408,5409..5415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5423..5439
                     /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     5447..5463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5447..5463
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5459..5907,1..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(5479..5496)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5479..5495)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5488..5510)
                     /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 tccttagacc atttagatgc agcagaggca
      961 gcctaccaga tcgagagcat catcaaaaca gcaactgaca ctgtgaagag tgagattagc
     1021 gctgaacttg gtgtggttcc tagcttaaac gcagttgaaa caggcgcgag ttctaacact
     1081 gaaccagaag aagccataca aactcgcaca gtgataaatc agcacggcgt gtccgagact
     1141 ttagtggaga attttctcag tagggcagct ttagtgtcaa agagaagttt cgaatataaa
     1201 aatcatacct cgtctaaagc tcggacagac aagaactttt tcaaatggac aattaatacc
     1261 aagtcctttg tacagttaag aagaaagcta gaattgttta cataccttag atttgatgct
     1321 gaaatcacca tactcacaac tgtggcagtg aatggtagta gtaacagcac atacatgggt
     1381 cttcctgact tgacacttca agcaatgttc gtaccaactg gtgctcttac tccagaaaag
     1441 caggattcat tccattggca gtcaggtagt aatgctagtg tattctttaa aatttctgat
     1501 cccccagcca gaatgaccat accttttatg tgcattaact cagcatactc agttttttat
     1561 gatggctttg ctggatttga gaaaagtggt ctgtatggaa taaatccagc tgacaccatc
     1621 ggcaacttat gcgtcagaat agtgaatgaa caccaaccaa ttggtttcac agtaaccgtc
     1681 agggtttaca tgaaacctaa acacataaaa gcatgggcac cgcgaccacc acgaacccta
     1741 ccatacatga gcattgcaaa tgcaaattac aggggtaaag atagagcacc aaatgccctc
     1801 aatgccataa ttggcaatag agagagtgtc aaaaccatgc cccacaatat agtgaccact
     1861 cccgggcacc atcatcacca ccatcccggg taataggaat tcatcgtgac tgactgacga
     1921 tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     1981 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     2041 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     2101 ataattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2161 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2221 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2281 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2341 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2401 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2461 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2521 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2581 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2641 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2701 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2761 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2821 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2881 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2941 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3001 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3061 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3121 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3181 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3241 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3301 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3361 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3421 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3481 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3541 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3601 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3661 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3721 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3781 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3841 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3901 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3961 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4021 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4081 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4141 cgcatctgtg cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct
     4201 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     4261 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     4321 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     4381 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     4441 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     4501 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     4561 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     4621 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     4681 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     4741 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     4801 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     4861 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     4921 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     4981 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     5041 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     5101 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     5161 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     5221 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5281 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5341 aacgcaatta atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5401 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5461 gaccatgatt acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     5521 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     5581 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt
     5641 tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga
     5701 ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta
     5761 caccaacgtg acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac
     5821 gggttgttac tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg
     5881 aattattttt gatggcgttg gaattag
//
