LOCUS       sequence-403580-        5874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of VP1 protein from rhinovirus A89 (RV-A89). 
            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 5874)
  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 5874)
  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_RV-A89_VP1
FEATURES             Location/Qualifiers
     source          1..5874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(182..187,188..203,204..210)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    218..234
                     /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             257..910
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     868..890
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             917..934
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1835..1852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1923..1945)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2090..2108)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2176..2280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2281..2349,2350..3141)
                     /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(2499..2518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3312..3900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3801..3820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4054..4071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4144..4221
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4222..5304
                     /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(4241..4260)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5353..5358,5359..5376,5377..5383)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5391..5407
                     /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     5415..5431
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5415..5431
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5427..5874,1..80)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5447..5464)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5447..5463)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5456..5478)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
       61 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
      121 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
      181 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
      241 acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca
      301 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
      361 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
      421 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
      481 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
      541 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
      601 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
      661 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
      721 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
      781 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
      841 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
      901 tcctccaaaa tcggatctgg ttccgcgtgg atccaaccca gttgaaaatt atatagatag
      961 tgtattaaat gaagttcttg tggtgccaaa tatccaacct agcacatctg tgtcaagtca
     1021 tgcagcgcct gcattggatg ctgcggaaac cggacacacc agctctgttc aacctgaaga
     1081 tatgattgaa actagatatg ttataactga tcaaacaagg gatgaaacaa gtattgagag
     1141 tttcttaggt aggtcagggt gtatcgctat gatagaattt aatacaagta gtgataaaac
     1201 tgaacatgat aaaattggta aaggattcaa aacatggaag gttagtcttc aagaaatggc
     1261 acaaatcaga agaaaatatg aattattcac atatacaaga tttgattcag agataacaat
     1321 agtcactgca gccgcagctc aaggaaatga tagtggacat atagtattgc aatttatgta
     1381 tgtaccccca ggagcacctg tccccgaaaa acgtgatgat tacacatggc aatcaggaac
     1441 aaatgcatct gtgttctggc aagaaggaca accatacccc agattcacaa tcccttttat
     1501 gagcattgca tcagcctatt acatgtttta tgatggttat gatggtgata gtgcagcatc
     1561 aaaatacggt tctgtagtca ctaatgatat gggaaccata tgtgttagaa tagtgacatc
     1621 caaccaaaaa cacgattcaa atattgtgtg ccgcatttac cacaaggcca aacatataaa
     1681 agcatggtgt cctcgcccac caagggctgt tgcctatcaa cacacacact caaccaatta
     1741 cataccatcc aatggtgagg ccacaactca gattaaaacc agacctgatg tttttaccgt
     1801 tacaaacgtc ggaccatcta gtatgtttcc cgggcaccat catcaccacc atcccgggta
     1861 ataggaattc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa
     1921 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     1981 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     2041 acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg cctcgtgata
     2101 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     2161 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2221 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2281 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2341 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2401 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2461 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2521 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2581 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2641 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2701 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2761 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2821 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2881 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2941 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3001 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3061 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3121 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3181 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3241 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3301 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3361 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3421 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3481 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3541 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3601 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3661 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3721 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3781 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3841 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3901 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3961 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4021 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4081 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat
     4141 tccgacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     4201 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     4261 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     4321 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     4381 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     4441 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     4501 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     4561 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     4621 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     4681 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     4741 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     4801 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     4861 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     4921 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     4981 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     5041 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     5101 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     5161 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     5221 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     5281 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     5341 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     5401 ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg gccgtcgttt
     5461 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5521 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5581 tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg
     5641 aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc
     5701 agatgcacgg ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc
     5761 cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg
     5821 aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttgga atta
//
