LOCUS       sequence-254053-        7386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inducible dual expression of NS3 protease along with its cofactor 
            NS4A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7386)
  AUTHORS   Lee H, Zhu T, Patel K, Zhang YY, Truong L, Hevener KE, Gatuz JL, 
            Subramanya G, Jeong HY, Uprichard SL, Johnson ME
  TITLE     High-throughput screening (HTS) and hit validation to identify small
            molecule inhibitors with activity against NS3/4A proteases from 
            multiple hepatitis C virus genotypes.
  JOURNAL   PLoS One. 2013 Oct 9;8(10):e75144. doi: 
            10.1371/journal.pone.0075144. eCollection 2013.
  PUBMED    24130685
REFERENCE   2  (bases 1 to 7386)
  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: "PLoS One.";
            date: "2013-10-9"; volume: "8(10):e75144. doi"; pages: " 
            10.1371/journal.pone.0075144. eCollection 2013"
COMMENT     Sequence Label: pETDuet-1 Hepatitis C Virus NS3/NS4A
FEATURES             Location/Qualifiers
     source          1..7386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(571..1362,1363..1431))
                     /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     1194..1213
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1432..1524)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1605..2193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2094..2113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2347..2364
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2379..2518
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2444..2463)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2604..2626
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2620..2811)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3383..4465)
                     /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     4427..4446
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4466..4543)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4749..4768
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4856..4874
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4875..4899
                     /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)."
     RBS             4914..4936
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        5192..5210
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5211..5235
                     /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             5281..5298
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7207..7251
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(7289..7307)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7303..7350
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt
      481 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
      541 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
      601 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
      661 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
      721 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
      781 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
      841 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
      901 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
      961 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     1021 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     1081 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     1141 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     1201 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     1261 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     1321 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     1381 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     1441 ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     1501 aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg
     1561 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1621 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1681 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1741 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1801 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1861 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1921 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1981 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2041 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2101 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2161 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2221 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2281 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2341 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     2401 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     2461 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     2521 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2581 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2641 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     2701 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     2761 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     2821 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     2881 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     2941 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     3001 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     3061 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     3121 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     3181 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     3241 ggtcctcaac gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct
     3301 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     3361 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3421 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     3481 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     3541 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     3601 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     3661 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     3721 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     3781 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     3841 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     3901 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     3961 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     4021 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     4081 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     4141 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     4201 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     4261 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     4321 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     4381 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     4441 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     4501 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     4561 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     4621 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     4681 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     4741 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     4801 ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata
     4861 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     4921 aactttaaga aggagatata ccatgggtag cacttgggta ctggtaggtg gtgtgctggc
     4981 tgcgctggcg gcttactgcc tgactacggg ttctgttgta atcgtaggtc gcatcatcct
     5041 gtccggccgt ccggcaattg tcccagatcg tgaactgctg tatcaggaat tcgatgaaat
     5101 ggaagaaggt aagaagaaat aagcttgcgg ccgcataatg cttaagtcga acagaaagta
     5161 atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga
     5221 gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga tatacatatg
     5281 caccaccatc atcaccatgc accgattacc gcgtattctc agcaaacgcg cggtctgctg
     5341 ggctgtatta tcacgtccct gactggtcgt gacaaaaacc aggtggaagg cgaagtgcag
     5401 gtcgtttcta ctgcaaccca gtccttcctg gccacctgcg taaacggcgt ctgctggacc
     5461 gtttaccacg gtgcgggcag caaaaccctg gccgcgccga aaggcccaat tactcagatg
     5521 tataccaacg tagatcagga tctggttggt tggccgaaac cgcctggtgc acgctccctg
     5581 accccgtgta cctgcggctc ttccgacctg tatctggtca cgcgtcatgc ggatgtaatc
     5641 ccggttcgcc gtcgcggtga ctcccgtggt agcctgctgt ctccgcgtcc tgttagctac
     5701 ctgaaaggca gctccggcgg tccgctgctg tgcccgtttg gccacgcagt tggtatcttc
     5761 cgtgcagccg tttgcacgcg tggcgttgcc aaagccgttg atttcgttcc ggtggaatct
     5821 atggaaacca ccatgcgctc tcctgtgttt accgataaca gctccccacc ggcagtgccg
     5881 caatctttcc aggttgcgca cctgcacgca ccaaccggca gcggtaaaag caccaaagtg
     5941 ccggcggcct acgcggccca gggttacaaa gtactggttc tgaatccgtc cgttgcagcg
     6001 acgctgggtt tcggtgcgta tatgtccaaa gctcacggca tcgaccctaa catccgcact
     6061 ggtgtgcgca ccattaccac cggcgcaccg gtgacctatt ccacctacgg caaattcctg
     6121 gcggatggtg gttgctccgg tggcgcatac gatatcatca tttgcgatga gtgtcactct
     6181 actgacagca ctaccatcct gggtattggc accgtgctgg atcaggcgga aacggctggt
     6241 gctcgtctgg tggtactggc taccgcgact ccgccgggta gcgttaccgt accgcacccg
     6301 aacatcgaag aggttgcgct gtctaatacc ggcgaaatcc cgttctatgg caaggccatc
     6361 ccgattgaag ccattcgcgg cggtcgccat ctgatcttct gtcactccaa gaaaaaatgt
     6421 gacgaactgg cggctaaact gagcggtctg ggtatcaacg cagtggcgta ctaccgtggt
     6481 ctggacgtgt ctgttattcc gaccatcggt gacgtcgtcg tagttgctac ggacgcactg
     6541 atgaccggct acaccggtga cttcgactct gtgatcgata gcaacacctg cgtaacccag
     6601 actgtggatt tctctctgga cccgaccttt actatcgaaa ccactactgt gccgcaggac
     6661 gctgttagcc gctctcaacg tcgtggccgt accggtcgcg gccgtcgtgg catttaccgt
     6721 tttgtcactc caggtgaacg tccgagcggc atgttcgact cctctgttct gtgcgagtgc
     6781 tacgacgctg gctgcgcgtg gtacgaactg actcctgctg agactagcgt tcgtctgcgt
     6841 gcctacctga acaccccggg tctgccagtg tgtcaggacc acctggaatt ctgggaatcc
     6901 gtttttaccg gcctgactca catcgatgcg cacttcctgt ctcagactaa acaagctggc
     6961 gataactttc cgtacctggt tgcatatcag gctactgtat gtgctcgtgc gcaggcacca
     7021 ccgccgtcct gggaccagat gtggaaatgt ctgatccgtc tgaaaccgac cctgcacggc
     7081 ccgaccccgc tgctgtatcg tctgggtgct gtccagaacg aggttactct gacgcatcct
     7141 atcaccaagt acatcatggc ttgcatgagc gctgacctgg aggttgttac ttaactcgag
     7201 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
     7261 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     7321 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     7381 aatggg
//
