LOCUS Exported 4623 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Coexpression construct of Nsp7 with an N-terminal His-tag and Nsp8. ACCESSION . VERSION . KEYWORDS pCDFDuet-nsp7-nsp8 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4623) AUTHORS Chen J, Malone B, Llewellyn E, Grasso M, Shelton PMM, Olinares PDB, Maruthi K, Eng ET, Vatandaslar H, Chait B, Kapoor T, Darst SA, Campbell EA TITLE Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex. JOURNAL bioRxiv. 2020 Jul 13. doi: 10.1101/2020.07.08.194084. PUBMED 32676607 REFERENCE 2 (bases 1 to 4623) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4623 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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 100..117 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 157..174 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 181..198 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 199..222 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" promoter 537..555 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 556..580 /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)." primer_bind complement(1242..1260) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1256..1303 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(1474..2265) /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" promoter complement(2266..2357) /gene="bla" /label=AmpR promoter CDS complement(3418..4500) /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 4462..4481 /label=LacI-R /note="LacI, reverse primer" promoter complement(4501..4578) /gene="lacI" /label=lacI promoter ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt 61 tgtttaactt taataaggag atataccatg ggcagcagcc atcaccatca tcaccacagc 121 caggatccga attcgaatgt gcagggacat cttcccctcg taccacgagg ttcaaagctt 181 catcatcatc atcatcatct ggaagttctg ttccaggggc ccgtcgacat gagtaagatg 241 agcgatgtta agtgtacgtc agttgttctt ctgtctgtac ttcagcagct tcgtgttgaa 301 tcatcgtcga agctgtgggc ccagtgcgta cagttacaca acgacatttt gctggcgaag 361 gatacaacgg aagcctttga gaagatggtc tcattacttt cggtccttct tagtatgcag 421 ggggccgtag acatcaataa gttatgtgaa gaaatgcttg acaaccgtgc aacgttacag 481 taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat tgaaattaat 541 acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta 601 agtataagaa ggagatatac atatggcgat tgcgtcagag ttctcaagtc ttcctagcta 661 cgcagccttt gcaaccgctc aggaagcata cgagcaagcg gttgcgaatg gcgacagcga 721 ggtcgtatta aaaaaattga aaaaaagcct taacgtagcc aaatccgaat tcgaccgcga 781 cgcagcgatg cagcgcaaat tagagaagat ggccgatcag gccatgacac agatgtataa 841 acaagcacgc agtgaagata agcgtgcaaa ggttacttct gcgatgcaaa caatgctttt 901 tacgatgttg cgtaaattag acaatgacgc gttaaacaac atcattaata atgcccgcga 961 tggatgcgta cctctgaata ttattccgtt gacaaccgcc gcgaaattaa tggtagtgat 1021 tcctgactac aatacctata aaaacacctg cgacggaaca acgtttacat acgcctcagc 1081 actgtgggag attcagcagg tggtggacgc tgactctaaa atcgtacagt tgtcagaaat 1141 tagtatggac aatagtccta accttgcttg gccacttatt gtcacagctc ttcgtgcaaa 1201 ttccgcggtc aaattgcagt aattaaccta ggctgctgcc accgctgagc aataactagc 1261 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt 1321 tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac 1381 ataagcggct atttaacgac cctgccctga accgacgacc gggtcatcgt ggccggatct 1441 tgcggcccct cggcttgaac gaattgttag acattatttg ccgactacct tggtgatctc 1501 gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc 1561 ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag 1621 gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct 1681 gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg 1741 cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg 1801 atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt 1861 cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc 1921 attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat 1981 gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga 2041 agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac 2101 ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga 2161 gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac 2221 ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatactct tcctttttca 2281 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 2341 ttagaaaaat aaacaaatag ctagctcact cggtcgctac gctccgggcg tgagactgcg 2401 gcgggcgctg cggacacata caaagttacc cacagattcc gtggataagc aggggactaa 2461 catgtgaggc aaaacagcag ggccgcgccg gtggcgtttt tccataggct ccgccctcct 2521 gccagagttc acataaacag acgcttttcc ggtgcatctg tgggagccgt gaggctcaac 2581 catgaatctg acagtacggg cgaaacccga caggacttaa agatccccac cgtttccggc 2641 gggtcgctcc ctcttgcgct ctcctgttcc gaccctgccg tttaccggat acctgttccg 2701 cctttctccc ttacgggaag tgtggcgctt tctcatagct cacacactgg tatctcggct 2761 cggtgtaggt cgttcgctcc aagctgggct gtaagcaaga actccccgtt cagcccgact 2821 gctgcgcctt atccggtaac tgttcacttg agtccaaccc ggaaaagcac ggtaaaacgc 2881 cactggcagc agccattggt aactgggagt tcgcagagga tttgtttagc taaacacgcg 2941 gttgctcttg aagtgtgcgc caaagtccgg ctacactgga aggacagatt tggttgctgt 3001 gctctgcgaa agccagttac cacggttaag cagttcccca actgacttaa ccttcgatca 3061 aaccacctcc ccaggtggtt ttttcgaagg ttaagtcagt tggggaactg cttaaccttc 3121 gatcaaacca cctccccagg tggttttttc gtttacaggg caaaagatta cgcgcagaaa 3181 aaaaggatct caagaagatc ctttgatctt ttctactgaa ccgctctaga tttcagtgca 3241 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc 3301 atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc 3361 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca 3421 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 3481 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac 3541 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac 3601 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca 3661 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc 3721 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc 3781 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact 3841 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca 3901 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg 3961 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa 4021 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt 4081 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc 4141 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg 4201 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc 4261 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga 4321 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc 4381 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga 4441 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac 4501 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 4561 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga 4621 aat //