LOCUS Exported 7386 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION inducible dual expression of NS3 protease along with its cofactor NS4A. ACCESSION . VERSION . KEYWORDS pETDuet-1 Hepatitis C Virus NS3/NS4A 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7386 /organism="synthetic DNA construct" /mol_type="other DNA" 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(571..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 //