LOCUS       sequence-420353-        5868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes EV-D68 MO/14/18947 3ABCD 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 5868)
  TITLE     Differential Inhibition of Intra- and Inter-molecular Protease 
            Cleavages by Antiviral Compounds
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5868)
  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            "Unpublished"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_3ABCD_C147R
FEATURES             Location/Qualifiers
     source          1..5868
                     /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(909..926)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3216..3242)
                     /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(3216..3242)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3250..3711
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     3638..3655
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(3761..3780)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3762..3780)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3787..3804)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3787..3803)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3796..3818)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3944..4399
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4031..4050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4241..4262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4468..4487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4587..4609
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4647..4665)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4733..4837
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4838..4906,4907..5698)
                     /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(5056..5075)
                     /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 tttttcagtc
      901 agatctcagt ggtggtggtg gtggtgctcg aggaacgaat ctaaccattt ccgtctaaga
      961 ctagagtatg caggtagtgt caatgctctt cccacaggca cacttctgat ttttctgcaa
     1021 aattcattat aagcctcctc tccattatgc catgcgaggt agcacaaaga cctaacatga
     1081 tcctgtgtgt ttctgggatc tttagtccat ctaatagatt catgtatctc tttcattggc
     1141 atcactggat gtatgagaaa ggggtattga tcatctgctc tgaaatatct ttttaagaaa
     1201 gttacatttt cccaatttgt gtcaataaaa ctggttcctt tgtctgctgg tgtcattact
     1261 aatccatact gtttacctgc ttctgccagc aaacctggat caatcttatg tgggtagcta
     1321 gcaataacat catccccata ggcaatcatt ttgaactggt ccaggtctat gcctttgtaa
     1381 accctaatta ataaagttct tatgattata ttgttgatca tagtgttgaa tatgctggtg
     1441 cctgaagaac cagagggcat tccaccatta actatgtatt ttttgtcttt atataaatgt
     1501 actgaatgac acaaataatc tataaaagac gtttgatgtg tgtaacctaa cttaattaga
     1561 actttcttta aacaggcaaa ccacactggt gacaaactag catcataacc agtgtagtca
     1621 aaagcaaaga tttctccatc taacaaaata gggatttttg accaaaatat atctggatca
     1681 caaccaactg cactaccagt tgctgtacct ggattgttgt gaaatgtggc gtaaaggttt
     1741 ccaaaggcaa ctctcatagc aactgagtca ttcaaggaac tggcctcaat caggcgtgat
     1801 ttcccttttt caactttttc tcttgatcta agctcatctt ttacaaaggt tacaaaaggt
     1861 aggtcaactc catatttctc taacattttt gtcatttcac tggtgtctct agtatgtcta
     1921 ttaaatatat cccttttctt cttcccttgt agtaagtaag ggaatcctgc actagtagtt
     1981 aagtctaatg cctcaaggcc atccattcca tacatggcac tttccagggg tatgggatcc
     2041 acactgatgt ctaatggttc taaacacccc acataatgat ccactgcctc ttccatgtac
     2101 tcatccatta acataatttt gttacctgtg tactttgaga aaatggcctc ctcgaaatct
     2161 gttttaagcc taggatcttt tggattgaga actgctggtt cctttgaacc ttcaaaaact
     2221 tgatggaaaa cactaggttg gagtttagtc tttgccggtg cgttaatgca caccccactc
     2281 ttttcactac taactatctc accttgtgta tcggaaaagt aagagtgtag tagcattgct
     2341 gcaaatcctt gagctccatt tccacctaca tgtattccta tcaccttacc tgtagttgtc
     2401 accacaccac cacgctggcc agctcttgtt gggaagttat acattaaaat gcggtgcgtc
     2461 ggtgtaccac ctaggttcaa gaagccataa ttggtgactt gtccaactgg gatatacata
     2521 tttgggaatt ttgatgtatg tacgctaagc acagcgtcat tgtaatcatc ctcatatctg
     2581 ggcagaaaat gtctgatatc tctaaatttt tgattacggt ctaatttgac tatggttatc
     2641 tctaagtttg tatcagtcaa gtctctaagt gcacacgcat ctaaaacttt agtctctaca
     2701 tcattaatgt aaatggtttc tccaacagat gcgtgtgtgg ggatgaccgc taccctatca
     2761 tatacaccca gcatggtgaa ctcacccttt tcagtccttg caatgacggt atttttcttc
     2821 attatggctt gtgcaaaatc gaaccctggt ccttgcactt tagctgttct gagagagggt
     2881 actttaggtt taggattggg gattcctgtg tatggaccct gaatgccagc aaaaagtttg
     2941 tatataacat aaactacacc agctattgat acaaaggtgg caatggcttg tagagtaata
     3001 aaagctctac taatgtgttt ttctactatt agctcatttg atgggtgtac tacaatccat
     3061 cctttctttt ggcaataatc cctaacttct tgagaatcca ctgacctaag aaggtcattt
     3121 atagcatcag gagctggtgt atctggggtg actgaaattt tgatctcttt aaactgtgga
     3181 ggcccgaatt cggatcccat atgggtggtg gccagggcgt agtcgggcac gtcgtagggg
     3241 taggccatat tatcatcgtg tttttcaaag gaaaaccacg tccccgtggt tcggggggcc
     3301 tagacgtttt ttaacctcga ctaaacacat gtaaagcatg tgcaccgagg ccccagatca
     3361 gatcccatac aatggggtac cttctgggca tccttcagcc ccttgttgaa tacgcttgag
     3421 gtgagccatt tgactctttc cacaactatc caactcacaa cgtggcactg gggttgtgcc
     3481 gcctttgcag gtgtatctta tacacgtggc ttttggccgc agaggcacct gtcgccaggt
     3541 ggggggttcc gctgcctgca aagggtcgct acagacgttg tttgtcttca agaagcttcc
     3601 agaggaactg cttccttcac gacattcaac agaccttgca ttcctttggc gagaggggaa
     3661 agacccctag gaatgctcgt caagaagaca gggccaggtt tccgggccct cacattgcca
     3721 aaagacggca atatggtgga aaataaccgg aattaattcg ccctatagtg agtcgtatta
     3781 caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     3841 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     3901 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc
     3961 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4021 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4081 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4141 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4201 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4261 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4321 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4381 atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4441 cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4501 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4561 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4621 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4681 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4741 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4801 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4861 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4921 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4981 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5041 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5101 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5161 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5221 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5281 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5341 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5401 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5461 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5521 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5581 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5641 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5701 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5761 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5821 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
