LOCUS       sequence-420359-        4961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Poliovirus type I Mahoney 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 4961)
  TITLE     Differential Inhibition of Intra- and Inter-molecular Protease 
            Cleavages by Antiviral Compounds
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4961)
  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            "Unpublished"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_polio_VP12A_C109R
FEATURES             Location/Qualifiers
     source          1..4961
                     /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(908..925)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1976..1999)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2309..2335)
                     /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(2309..2335)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2343..2804
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     2731..2748
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2854..2873)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2855..2873)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2880..2897)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2880..2896)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2889..2911)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3037..3492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3124..3143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3334..3355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3561..3580)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3680..3702
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3740..3758)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3826..3930
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3931..3999,4000..4791)
                     /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(4149..4168)
                     /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 ttttcagtca
      901 gatctcagtg gtggtggtgg tggtgctcga gttgttccat ggcttcttct tcgtaggcat
      961 acaagtctct aatgtctgaa aatgcaacca acccttcgcc accagcagta atgatcccta
     1021 tcaccccgtg gtgacatctg agtatgccac cgcgatcccc tggagatgcg aatccatggc
     1081 caatgagcat atgggactgg tacctagctg ggtaatagtt attagcctcc atgtactgga
     1141 acgttgggcc aacgaaggat actgggtagt atttccttct agactcgcag tagtacaccc
     1201 ctgcgttgca attgcacctt gcgattgaat cggtgccctg ggctcttgat tctgtgacta
     1261 agaggtctct actccacatg acgttcactg cgttttgcaa atcatcctga gtggccaagt
     1321 ggtagttgca aattttgtaa cctgcagtgt acaccgcttt gttttggtgt ccgaatccat
     1381 atgtggtcag atccttggtg gagaggggtg taagcgtacc atccttgtaa tccactccag
     1441 ggccgtagta cgccactgcc ctcggtggac gcgggcacca gactctgatg tgtttgggtt
     1501 ttagatacac tctgattttg gaggtgacct tggtcgggtt gtgatcattg actactctaa
     1561 cagccaaaat accgaagtca tttagagatg ctgcaccata aagggagtca cctagtgctg
     1621 ccgactggtc cttcagtggt actttggaaa aaccgtcgta aaagtgtgaa taggcgttcg
     1681 aaataccaac atacggtacc gagatccggg ctggagctgt tccgtaggtg taaaagattg
     1741 atggatttga tgaggtttgc catgtgtagt cgtcccattt ctcgggcact ggagcgcctg
     1801 gtggtacgta cataatttgg tacacttgat ttaaggcatg cccattgtta gtctcagtga
     1861 aatttgcagt aaccacaaag gtaagttcca tatcaaatct agaataggtg aagaactcca
     1921 atttcctccg taactggaca gtatctttat aagtgatctt ccacactgca aatagcttat
     1981 catcatcatc tttgtagtct gggttatcca cggtcataat ggtcacgcat gcaccccgcg
     2041 cgaagaaaga ctctatgcta gactctgacc ttgacctatg ttgtacaaca tgtctggttt
     2101 gcactgtatc agaagggact agtggatttg tggccccagt ttccactgcg gtgagtgccg
     2161 gaatttcctt ggagtgtgtt ggtccactgg cttcagtgtt tgggagagcg tctctagatg
     2221 ttgccgcccc caccgtttca cggactgtgt tgtcaatcat gctttcaagc atctgaccta
     2281 acccggatcc catatgggtg gtggccaggg cgtagtcggg cacgtcgtag gggtaggcca
     2341 tattatcatc gtgtttttca aaggaaaacc acgtccccgt ggttcggggg gcctagacgt
     2401 tttttaacct cgactaaaca catgtaaagc atgtgcaccg aggccccaga tcagatccca
     2461 tacaatgggg taccttctgg gcatccttca gccccttgtt gaatacgctt gaggtgagcc
     2521 atttgactct ttccacaact atccaactca caacgtggca ctggggttgt gccgcctttg
     2581 caggtgtatc ttatacacgt ggcttttggc cgcagaggca cctgtcgcca ggtggggggt
     2641 tccgctgcct gcaaagggtc gctacagacg ttgtttgtct tcaagaagct tccagaggaa
     2701 ctgcttcctt cacgacattc aacagacctt gcattccttt ggcgagaggg gaaagacccc
     2761 taggaatgct cgtcaagaag acagggccag gtttccgggc cctcacattg ccaaaagacg
     2821 gcaatatggt ggaaaataac cggaattaat tcgccctata gtgagtcgta ttacaattca
     2881 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     2941 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3001 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     3061 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3241 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3301 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3481 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3541 catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3601 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3661 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3721 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3781 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3841 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3901 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3961 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4021 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4081 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4141 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4201 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4261 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4321 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4381 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4441 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4501 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4561 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4621 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4681 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4741 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4801 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4861 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4921 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
