LOCUS       sequence-420360-        4948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes EV-A71 4643 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 4948)
  TITLE     Differential Inhibition of Intra- and Inter-molecular Protease 
            Cleavages by Antiviral Compounds
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4948)
  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            "Unpublished"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_EVA71_VP12A_C110A
FEATURES             Location/Qualifiers
     source          1..4948
                     /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(2296..2322)
                     /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(2296..2322)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2330..2791
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     2718..2735
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2841..2860)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2842..2860)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2867..2884)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2867..2883)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2876..2898)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3024..3479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3111..3130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3321..3342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3548..3567)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3667..3689
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3727..3745)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3813..3917
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3918..3986,3987..4778)
                     /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(4136..4155)
                     /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 cttctccatg gcttcatcat ccaaccacag
      961 aagatccctc acatcggcga accccaccag gccgtttccc ccggtggaaa ctatccctac
     1021 gacgccatgt tggcatctaa gaatgccacc ggcatcccct ggttccgaat gacccactgc
     1081 aagcatgaga tgtgactggt atctagctgg ataatactcg ctggcctcca cgaagatcaa
     1141 actgggtttc gagaaactga ccggatagtg ttttctcatt gagttacaat aatacactcc
     1201 tgtctggcaa ttgcaacgag caatcgtgtc acaaccttga gcagtggtag atgatacgag
     1261 caagtcgcgg gagctgtctt cccaaacaag gtttgcccag tcattatgag tagcaagatg
     1321 gcggttaacc actctaaagt tgcccacgta gatagctccg gactgctgtc caaatttccc
     1381 gagggtggtg attgctgtgc gactggcacc agttggttta ataaaattac cagcataatt
     1441 tgggttggct ttgaatagat agttctggtt acgcattggg cgaggtatcc acgccctgac
     1501 gtgcttcatc ctcatgtaaa tcctgatcac caatgggtac ttggacttcg aggtgcctac
     1561 agtccgcact gagaacgtac ccatcatgtt gtttgggcat gccccgtatt caaggtcttt
     1621 ctcctgtttg tgttcaccga atgtgggata cccgtcataa aaccattgat aggcgctcgc
     1681 aggtgacatg aatggaacag aaacctgtgc tggggggtct gacagcttca caaaaactga
     1741 gggattagtg gcagtttgcc atgcgagaga ttctctggag tctggcttgg gggctccggg
     1801 tggtacaaac atgtattgga gcaattgcgg gacaacttgc ccggtagggg tgcatgcaac
     1861 aaaggtgaac tctgcgtcaa aacgcatgta ggtgaacagc tccacctttc tacgcatttg
     1921 cgcgtaacct gttatgtcta tgtcccagtt tgcgtaccca ttcgggttgg ttgtgccttc
     1981 aagagggagg tctatctctc caactaatcc tgctctgctg aagaagctat caagagtggt
     2041 ctcagctgta ctatgtgaat taagaacaca ccgtgtctca atcatactct catcactagc
     2101 atttgatgat gctccaattt cagcggcttg gagtgctgga actttaccag tatctaatcg
     2161 gtggctgctt acctgtgtgt cttggccggt aggtgccggt agagctcggg tgagggctct
     2221 gctcacactg tcccctatag aactctcaat cacatctgcc accctatctc cggatcccat
     2281 atgggtggtg gccagggcgt agtcgggcac gtcgtagggg taggccatat tatcatcgtg
     2341 tttttcaaag gaaaaccacg tccccgtggt tcggggggcc tagacgtttt ttaacctcga
     2401 ctaaacacat gtaaagcatg tgcaccgagg ccccagatca gatcccatac aatggggtac
     2461 cttctgggca tccttcagcc ccttgttgaa tacgcttgag gtgagccatt tgactctttc
     2521 cacaactatc caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta
     2581 tacacgtggc ttttggccgc agaggcacct gtcgccaggt ggggggttcc gctgcctgca
     2641 aagggtcgct acagacgttg tttgtcttca agaagcttcc agaggaactg cttccttcac
     2701 gacattcaac agaccttgca ttcctttggc gagaggggaa agacccctag gaatgctcgt
     2761 caagaagaca gggccaggtt tccgggccct cacattgcca aaagacggca atatggtgga
     2821 aaataaccgg aattaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
     2881 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2941 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3001 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3301 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3361 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3421 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
     3481 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca
     3541 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3601 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3661 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3721 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3781 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3841 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3901 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3961 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4021 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4081 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4141 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4201 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4261 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4321 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4381 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4441 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4501 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4561 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4621 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4681 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4741 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4801 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4861 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4921 accccgtaga aaagatcaaa ggatcttc
//
