LOCUS       sequence-312923-        4623 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coexpression construct of Nsp7 with an N-terminal His-tag and Nsp8.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 BT, Kapoor TM, Darst SA, 
            Campbell EA
  TITLE     Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 
            Replication-Transcription Complex.
  JOURNAL   Cell. 2020 Sep 17;182(6):1560-1573.e13. doi: 
            10.1016/j.cell.2020.07.033. Epub 2020 Jul 28.
  PUBMED    32783916
REFERENCE   2  (bases 1 to 4623)
  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"; doi: 
            "10.1016/j.cell.2020.07.033"; journalName: "Cell"; date: 
            "2020-09-17- 17"; volume: "182"; issue: "6"; pages: "1560-1573.e13"
COMMENT     Sequence Label: pCDFDuet-nsp7-nsp8
FEATURES             Location/Qualifiers
     source          1..4623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
//
