LOCUS       sequence-307877-        7964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 
            Nsp12 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7964)
  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 7964)
  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-Nsp12-HF
FEATURES             Location/Qualifiers
     source          1..7964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     674..694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    725..743
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    746..764
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     774..798
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3721..3744
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3757..3780
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3821..3838)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3827..4051
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4184..4203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4335..4382
                     /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             4390..5412
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4390..5412
                     /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    5542..5663
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5579..5598)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5633..5652
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5712..5728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5712..5728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5725..5747)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5736..5752
                     /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(5760..5766,5767..5784,5785..5790))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5805..5826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5943..5960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6114..6702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6194..6213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6873..7664,7665..7733))
                     /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     7496..7515
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7734..7838)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7913..7932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
       61 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
      121 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      661 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      721 gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt
      781 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      841 caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttgg taccgagctc
      901 ggatccggga tgtcagcgga tgcgcagtct tttcttaatc gggtttgcgg agtatctgcg
      961 gcccggctta ccccgtgcgg aactggcacc agtaccgatg tggtatacag ggccttcgac
     1021 atatataacg ataaggtcgc gggttttgca aagtttctca agactaattg ctgtcgcttt
     1081 caggaaaaag acgaagatga caatttgatc gattcctatt tcgtggttaa gagacatact
     1141 ttttctaact accaacatga agaaacaata tacaatctcc tcaaagattg tcccgccgtc
     1201 gccaaacacg acttttttaa gtttcgaatc gatggcgata tggtcccgca tatttctcga
     1261 cagcggctca ctaagtacac tatggcggac cttgtgtatg ctctccgcca ctttgacgag
     1321 gggaattgtg acacgcttaa agagatcctt gtaacctata actgctgtga tgatgactat
     1381 ttcaataaga aggactggta cgattttgtt gagaaccccg acatcctgcg cgtgtatgcc
     1441 aatctcggag agcgggtacg gcaagcgttg ctgaaaaccg tacaattctg tgacgcgatg
     1501 cgcaacgcag gaatcgtagg tgtgctcaca ctggacaatc aggatcttaa cggtaattgg
     1561 tacgattttg gagattttat acagactacc cctggctccg gtgtcccagt agtggattca
     1621 tattactcct tgctcatgcc catacttacc cttacgaggg ctttgacggc agagagtcat
     1681 gtagatactg accttacgaa gccgtacatt aaatgggacc tgttgaagta tgattttacg
     1741 gaagagaggt tgaagttgtt cgataggtac tttaaatact gggatcagac ttatcatcca
     1801 aattgcgtta attgcttgga tgaccgatgc attttgcact gtgcgaattt caacgtgctc
     1861 ttttccacgg ttttcccccc gacgtccttt ggaccactgg tcaggaaaat ttttgtcgac
     1921 ggtgtaccct ttgtggtctc cacaggatac cattttcgag agcttggagt cgtacacaat
     1981 caagatgtaa acctgcattc aagtcgcctc tcctttaaag aactccttgt ctatgcagcc
     2041 gatcctgcaa tgcacgcagc gtcagggaac ctcctcttgg acaaaaggac cacgtgtttc
     2101 tcagtcgcag cgttgacgaa taacgtggcg ttccagaccg taaagcctgg taatttcaac
     2161 aaagacttct atgatttcgc ggtcagtaaa gggttcttca aggaaggatc atcagttgag
     2221 cttaagcact ttttttttgc ccaagacggc aacgcagcaa tttccgatta tgactactat
     2281 cgatacaacc tgcctactat gtgtgacatt cgccagcttc ttttcgtcgt tgaagttgta
     2341 gataagtact ttgattgcta tgatggcggt tgtattaatg cgaaccaggt gatcgtcaac
     2401 aatctcgata aatctgccgg gtttcccttt aacaaatggg gcaaagcgag gttgtattac
     2461 gattctatgt cctacgagga tcaggatgcc ctcttcgcat acaccaagcg gaatgtgatt
     2521 cctaccatta cacaaatgaa cttgaaatac gccatatctg ccaaaaaccg cgctcgcact
     2581 gttgcaggtg tgtctatatg ctccacgatg actaataggc agtttcatca gaagttgctc
     2641 aaaagtatcg cagccactcg aggggcaacg gttgtgatag ggacgagtaa gttttatggc
     2701 ggctggcata atatgctgaa gaccgtgtat agcgatgttg agaatccaca cctcatgggt
     2761 tgggattatc cgaaatgcga tcgcgctatg cctaatatgc tcagaatcat ggcaagtctt
     2821 gtgttggctc ggaaacacac aacttgctgc tctctttctc ataggttcta tagacttgct
     2881 aacgaatgtg cccaggttct ctccgagatg gttatgtgtg gcggtagctt gtacgtaaag
     2941 ccgggaggga catcttctgg tgacgccacg actgcgtatg ctaattctgt attcaacatc
     3001 tgtcaggcag taactgcgaa cgttaacgcc ttgctcagca cagacggcaa caagatagcg
     3061 gataaatacg tgcgcaatct gcagcacagg ctgtatgagt gtttgtatcg caatcgagat
     3121 gtggacaccg acttcgtgaa tgagttttat gcttatctca gaaaacactt ttctatgatg
     3181 atactcagcg atgatgccgt ggtctgtttt aacagcactt acgcaagcca aggccttgtt
     3241 gcgagtatca aaaattttaa gagcgtgttg tattaccaaa ataatgtatt catgagcgag
     3301 gccaaatgct ggacagagac cgacctcaca aaggggccac acgagttttg ctcccagcac
     3361 acgatgctgg tcaaacaggg agatgactat gtatacctgc cgtatcccga tccgtctcgg
     3421 attctggggg cgggctgctt tgtcgatgac atagtgaaga cggacggcac acttatgatc
     3481 gaacggtttg tttcactggc aatagatgcg taccctttga ctaaacatcc caaccaggaa
     3541 tatgcggatg tgttccatct ttacttgcaa tatattagga agttgcacga cgagctgacg
     3601 gggcatatgc ttgatatgta ctcagtcatg cttacgaatg ataatacctc acgctattgg
     3661 gagccagaat tctatgaggc gatgtataca ccacacacag tgctgcaggc atcgggcgga
     3721 catcaccacc atcatcacca tcacgccgca gccgcagatt ataaagatga cgatgacaag
     3781 tagctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     3841 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3901 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3961 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     4021 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     4081 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4141 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4201 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4261 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4321 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     4381 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     4441 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     4501 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     4561 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     4621 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     4681 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     4741 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     4801 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     4861 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     4921 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     4981 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     5041 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     5101 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     5161 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     5221 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     5281 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     5341 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     5401 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5461 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5521 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5581 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5641 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5701 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5761 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5821 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5881 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5941 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6001 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6061 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6121 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6181 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6241 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6301 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6361 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6421 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6481 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6541 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6601 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6661 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6721 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6781 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6841 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6901 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6961 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7021 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7081 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7141 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7201 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7261 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7321 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7381 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7441 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7501 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7561 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7621 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7681 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7741 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7801 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7861 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     7921 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgt
//
