LOCUS       sequence-307127-        4558 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 4558)
  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 4558)
  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..4558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(484..1566)
                     /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     1528..1547
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1567..1644)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1690..1708
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1709..1733
                     /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             1789..1806
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1846..1863
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1870..1887
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1888..1911
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        2226..2244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2245..2269
                     /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(2931..2949)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2945..2992
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3163..3954)
                     /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(3955..4046)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4094..4558,1..274))
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 cggtaaaacg ccactggcag cagccattgg taactgggag ttcgcagagg atttgtttag
       61 ctaaacacgc ggttgctctt gaagtgtgcg ccaaagtccg gctacactgg aaggacagat
      121 ttggttgctg tgctctgcga aagccagtta ccacggttaa gcagttcccc aactgactta
      181 accttcgatc aaaccacctc cccaggtggt tttttcgttt acagggcaaa agattacgcg
      241 cagaaaaaaa ggatctcaag aagatccttt gatcttttct actgaaccgc tctagatttc
      301 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta
      361 attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
      421 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
      481 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
      541 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
      601 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
      661 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
      721 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
      781 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
      841 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
      901 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
      961 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     1021 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     1081 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     1141 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     1201 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     1261 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     1321 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     1381 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     1441 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     1501 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     1561 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     1621 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     1681 ttaggaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctgtag
     1741 aaataatttt gtttaacttt aataaggaga tataccatgg gcagcagcca tcaccatcat
     1801 caccacagcc aggatccgaa ttcgaatgtg cagggacatc ttcccctcgt accacgaggt
     1861 tcaaagcttc atcatcatca tcatcatctg gaagttctgt tccaggggcc cgtcgacatg
     1921 agtaagatga gcgatgttaa gtgtacgtca gttgttcttc tgtctgtact tcagcagctt
     1981 cgtgttgaat catcgtcgaa gctgtgggcc cagtgcgtac agttacacaa cgacattttg
     2041 ctggcgaagg atacaacgga agcctttgag aagatggtct cattactttc ggtccttctt
     2101 agtatgcagg gggccgtaga catcaataag ttatgtgaag aaatgcttga caaccgtgca
     2161 acgttacagt aatgcttaag tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc
     2221 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc catcttagta
     2281 tattagttaa gtataagaag gagatataca tatggcgatt gcgtcagagt tctcaagtct
     2341 tcctagctac gcagcctttg caaccgctca ggaagcatac gagcaagcgg ttgcgaatgg
     2401 cgacagcgag gtcgtattaa aaaaattgaa aaaaagcctt aacgtagcca aatccgaatt
     2461 cgaccgcgac gcagcgatgc agcgcaaatt agagaagatg gccgatcagg ccatgacaca
     2521 gatgtataaa caagcacgca gtgaagataa gcgtgcaaag gttacttctg cgatgcaaac
     2581 aatgcttttt acgatgttgc gtaaattaga caatgacgcg ttaaacaaca tcattaataa
     2641 tgcccgcgat ggatgcgtac ctctgaatat tattccgttg acaaccgccg cgaaattaat
     2701 ggtagtgatt cctgactaca atacctataa aaacacctgc gacggaacaa cgtttacata
     2761 cgcctcagca ctgtgggaga ttcagcaggt ggtggacgct gactctaaaa tcgtacagtt
     2821 gtcagaaatt agtatggaca atagtcctaa ccttgcttgg ccacttattg tcacagctct
     2881 tcgtgcaaat tccgcggtca aattgcagta attaacctag gctgctgcca ccgctgagca
     2941 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     3001 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     3061 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg
     3121 gccggatctt gcggcccctc ggcttgaacg aattgttaga cattatttgc cgactacctt
     3181 ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa
     3241 gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg
     3301 ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt
     3361 tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca
     3421 gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc
     3481 aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct
     3541 tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc
     3601 aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca
     3661 cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc
     3721 tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc
     3781 aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc
     3841 cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac
     3901 gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt
     3961 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4021 tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacg ctccgggcgt
     4081 gagactgcgg cgggcgctgc ggacacatac aaagttaccc acagattccg tggataagca
     4141 ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc
     4201 cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg
     4261 aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc
     4321 gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata
     4381 cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt
     4441 atctcggctc ggtgtaggtc gttcgctcca agctgggctg taagcaagaa ctccccgttc
     4501 agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagca
//
