LOCUS       sequence-422577-        4975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes EV-D68 MO/14/18947 VP12A with active site mutation optimized
            for expression in mammalian transcription/translation extracts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4975)
  TITLE     Differential Inhibition of Intra- and Inter-molecular Protease 
            Cleavages by Antiviral Compounds
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4975)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pT7CFE1_EVD68_VP12A_C107R
FEATURES             Location/Qualifiers
     source          1..4975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(789..836)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     832..850
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(907..924)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2323..2349)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(2323..2349)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2357..2818
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     2745..2762
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2868..2887)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2869..2887)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2894..2911)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2894..2910)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2903..2925)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3051..3506
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3138..3157)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3348..3369
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3575..3594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3694..3716
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3754..3772)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3840..3944
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3945..4013,4014..4805)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4163..4182)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcccgga tatagttcct
      781 cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat
      841 tgctcagcgg actagtgttt aaactttttt tttttttttt tttttttttt tttcagtcag
      901 atctcagtgg tggtggtggt ggtgctcgag ttgttccata gcatcagtat ctaaccatag
      961 caaattcctg atatcagtga aagctacaat tccaccccct cctgctgtaa gaagaccgat
     1021 taccccatgt ggacaaacta gtaaaccacc gcgatctcct gcttccgcag gaccaactgc
     1081 caaaagtaca ttggtctggt accttgctgg gtaatattca ttttgttcaa tccattgaat
     1141 ccctgggcct tcaaagcaaa tcgggtaact tctgtttttg tgcctacaat agtaaacccc
     1201 tgtgttgcac ttgcatcttg ctatttggtg ccttccatga gcagcaatgg gggtaaccaa
     1261 gacgtctgat tgccaatcca catagatagc tgactgtctc tcttctgtag tggccaagtg
     1321 atagttgatt attttaaaag accctacaaa aactcctccg aagcctggac cagtgttcac
     1381 tatattatga ggcatggttt tgacactgtc tctattgcca attatagcac tgagcgcatt
     1441 tggtgctctt tgtttacctt tgtaatttgc atttgcaata ctcatatatg gcagagttcg
     1501 tggtggtcgt ggtgcccatg cttttatgtg tttaggcttc atgtaaaccc taacggtcac
     1561 tgtgaaacca actggttggt gttcattcac tattctaaca cataagttac caatagtgtc
     1621 agctggattt attccataca gaccgttttt ctcaaatccg gcaaagccat cataaaaaac
     1681 tgagtatgct gagttaatgc acataaaagg tatggttatt ctggctgggg ggtcggagat
     1741 tttaaagaat acactagcat tactgcctga ctgccagtgg aatgagtcct gcttttctgg
     1801 ggtaagagca ccagtgggta caaacattgc ttgaagtgtc aagtcaggaa gacccacgta
     1861 tgtattatta ccactaccat tcactgctac agttgtgagt atagtgatct cagcatcaaa
     1921 tctaaggtat gtgaataatt ctaattttct tcttaactgt acaaaggatc tggtgttaat
     1981 tgtccatttg aaaaagttct tgtctgctcg tgctgtagac gaagtatgat ctttgtattc
     2041 aaaacttctc tttgatacca aagctgctct actgagaaaa ttctccacta gagtctcgga
     2101 tacaccgtgc tgatttatca ctgtgcgagt ttgtatggct tcttctggtt cagtgttaga
     2161 agttacacct gtttcaactg catttaagct agggaccaca ccaagttcag cattaatctc
     2221 acttttcaca gtgtcggtcg ctgttttgat gatgctctcg atctggtagg ctgcctctgc
     2281 tgcatgtaaa tggtctaggg atcccatatg ggtggtggcc agggcgtagt cgggcacgtc
     2341 gtaggggtag gccatattat catcgtgttt ttcaaaggaa aaccacgtcc ccgtggttcg
     2401 gggggcctag acgtttttta acctcgacta aacacatgta aagcatgtgc accgaggccc
     2461 cagatcagat cccatacaat ggggtacctt ctgggcatcc ttcagcccct tgttgaatac
     2521 gcttgaggtg agccatttga ctctttccac aactatccaa ctcacaacgt ggcactgggg
     2581 ttgtgccgcc tttgcaggtg tatcttatac acgtggcttt tggccgcaga ggcacctgtc
     2641 gccaggtggg gggttccgct gcctgcaaag ggtcgctaca gacgttgttt gtcttcaaga
     2701 agcttccaga ggaactgctt ccttcacgac attcaacaga ccttgcattc ctttggcgag
     2761 aggggaaaga cccctaggaa tgctcgtcaa gaagacaggg ccaggtttcc gggccctcac
     2821 attgccaaaa gacggcaata tggtggaaaa taaccggaat taattcgccc tatagtgagt
     2881 cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2941 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     3001 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     3061 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3121 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3181 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3241 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3301 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3361 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3421 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3481 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     3541 ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3601 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3661 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3721 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3781 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3841 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3901 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3961 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4021 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4081 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4141 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4201 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4261 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4321 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4381 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4441 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4501 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4561 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4621 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4681 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4741 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4801 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4861 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4921 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
