LOCUS       sequence-307832-        5417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 
            Nsp7 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5417)
  AUTHORS   Bresson S, Robertson N, Sani E, Turowski TW, Shchepachev V, 
            Kompauerova M, Spanos C, Helwak A, Tollervey D
  TITLE     Integrative vectors for regulated expression of SARS-CoV-2 proteins 
            implicated in RNA metabolism.
  JOURNAL   Wellcome Open Res. 2020 Nov 3;5:261. doi: 
            10.12688/wellcomeopenres.16322.1. eCollection 2020.
  PUBMED    33313418
REFERENCE   2  (bases 1 to 5417)
  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: "Wellcome 
            Open Res."; date: "2020-11-3"; volume: "5:261. doi"; pages: " 
            10.12688/wellcomeopenres.16322.1. eCollection 2020"
COMMENT     Sequence Label: pcDNA5-FRT-TO-Nsp7-HF
FEATURES             Location/Qualifiers
     source          1..5417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        97..476
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        477..680
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     631..651
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    682..700
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    703..721
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     731..755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1131..1154
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1167..1190
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1231..1248)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1237..1461
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1594..1613)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1745..1792
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             1800..2822
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1800..2822
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    2952..3073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2989..3008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3043..3062
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3122..3138)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3122..3138)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3135..3157)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3146..3162
                     /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)."
     promoter        complement(join(3170..3176,3177..3194,3195..3200))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3215..3236
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3353..3370)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3524..4112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3604..3623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4283..5074,5075..5143))
                     /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     4906..4925
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5144..5248)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5323..5342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
       61 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      121 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      181 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      241 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      301 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      361 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      421 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      481 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      541 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      601 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      661 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
      721 atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
      781 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta
      841 agcttggtac cgagctcgga tccgggatgt ctaagatgag tgatgtgaaa tgtacgtccg
      901 tggtgttgct ttctgtcctg caacagctta gagttgaatc tagcagtaag ctctgggcac
      961 aatgcgtgca actccacaat gatattctgt tggcgaaaga tactactgaa gccttcgaga
     1021 agatggtatc cttgttgtct gtccttttga gcatgcaagg cgcggttgac atcaacaagt
     1081 tgtgtgagga gatgctggat aaccgagcta cactgcaagc atcgggcgga catcaccacc
     1141 atcatcacca tcacgccgca gccgcagatt ataaagatga cgatgacaag tagctcgagt
     1201 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1261 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1321 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1381 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1441 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     1501 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1561 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1621 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1681 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1741 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     1801 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     1861 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     1921 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     1981 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     2041 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     2101 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     2161 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     2221 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     2281 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     2341 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     2401 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     2461 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     2521 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     2581 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     2641 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     2701 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     2761 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     2821 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     2881 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     2941 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3001 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3061 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3121 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3181 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3241 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3301 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3361 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3421 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3481 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3541 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3601 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3661 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3721 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3781 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3841 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3901 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3961 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4021 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4081 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4141 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4201 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4261 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4321 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4381 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4441 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4501 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4561 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4621 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4681 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4741 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4801 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4861 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4921 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4981 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5041 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5101 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5161 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5221 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     5281 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     5341 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     5401 gagcaaaatt taagcta
//
