LOCUS       sequence-307914-        6759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp14 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  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 6759)
  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-FH-Nsp14
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        172..551
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        552..755
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     706..726
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    757..775
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    778..796
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     806..830
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             927..950
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             963..986
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2648..2665)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2654..2878
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3011..3030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3162..3209
                     /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             3217..4239
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3217..4239
                     /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    4369..4490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4406..4425)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4460..4479
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4539..4555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4539..4555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4552..4574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4563..4579
                     /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(4587..4593,4594..4611,4612..4617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4632..4653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4770..4787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4941..5529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5021..5040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5700..6491,6492..6560))
                     /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     6323..6342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6561..6665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6740..6759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
       61 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      121 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      181 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      241 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      301 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      361 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      421 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      481 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      541 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      601 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      661 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      721 ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat agagatctcc
      781 ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc gcctggagac
      841 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc
      901 tagcgtttaa acttaagctt gagatggact ataaagatga cgatgacaag gccgcagccg
      961 cacatcacca ccatcatcac catcacggat ccgggatggc tgaaaatgtg actggtctct
     1021 tcaaagattg ctccaaggtg attacgggcc tgcatcccac ccaggccccc acacatcttt
     1081 cagtagacac gaaatttaaa actgaaggtc tttgtgttga catccctggc attccgaagg
     1141 atatgactta tcgccggctt atctccatga tggggtttaa gatgaattac caggtcaacg
     1201 ggtacccaaa catgttcatc acacgagagg aggcaattcg gcacgttaga gcatggattg
     1261 gttttgacgt tgagggatgc cacgccactc gagaggcggt cgggaccaac ttgccactcc
     1321 agttgggctt ctccaccggc gtcaatctgg tcgcagtacc cacaggttac gtggacacgc
     1381 ccaataacac agacttttca agagtaagcg ctaaaccccc acccggagat cagttcaaac
     1441 acttgatacc cctgatgtac aaaggtcttc cctggaacgt tgtccgaatc aaaatcgtcc
     1501 agatgctctc tgatacgctc aagaacctga gcgatcgagt cgttttcgta ctctgggcgc
     1561 atggtttcga gctgacctcc atgaagtact tcgtaaaaat agggcctgag agaacatgct
     1621 gtctgtgtga tcgaagagct acctgtttta gcaccgcttc cgatacatat gcttgctggc
     1681 atcattcaat aggtttcgat tacgtataca acccattcat gatcgatgtg cagcagtggg
     1741 ggttcactgg gaacctgcaa tcaaaccacg atctgtattg tcaagtacac ggtaatgcgc
     1801 acgtcgcttc atgtgatgcc atcatgacgc ggtgcctggc agtacatgag tgctttgtaa
     1861 agagggttga ttggacgata gagtatccta ttatcggaga cgaactcaaa ataaatgctg
     1921 cctgccgcaa agttcagcat atggttgtaa aagcggccct tctcgcggat aaatttccgg
     1981 ttttgcatga catcggaaat ccgaaagcaa taaagtgcgt cccgcaggca gacgtggaat
     2041 ggaaatttta tgatgcacaa ccttgcagtg ataaggccta taaaattgaa gaactgttct
     2101 acagctacgc cacccactct gataagttca cagacggagt ctgtcttttt tggaattgta
     2161 acgtcgatcg gtatccagct aattctattg tctgtcgatt tgacacgagg gttttgtcaa
     2221 acctgaacct tccaggatgt gatggaggtt ctttgtacgt caataaacac gcattccaca
     2281 cgcccgcctt tgataaatct gcattcgtaa atctcaagca gcttcccttc ttttactaca
     2341 gtgattcccc atgtgaatca catggcaagc aagttgtcag cgatatagac tatgtgcccc
     2401 ttaagtctgc aacctgcatc acgcgctgta atttgggagg tgcagtctgt cggcatcatg
     2461 cgaatgagta taggctttat ctcgacgcgt acaacatgat gatttcagcg gggttttcat
     2521 tgtgggtata taagcaattc gacacttata acttgtggaa cacatttacg cgcctccagg
     2581 cctaggagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc
     2641 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2701 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2761 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2821 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2881 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2941 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3001 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3061 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3121 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     3181 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     3241 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3301 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3361 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3421 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3481 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3541 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3601 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     3661 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3721 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3781 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3841 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     3901 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     3961 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     4021 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     4081 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4141 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4201 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     4261 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4321 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4381 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4441 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4501 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4561 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4621 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4681 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4741 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4801 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4861 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4921 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4981 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5041 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5101 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5161 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5221 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5281 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5341 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5401 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5461 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5521 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5581 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5641 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5701 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5761 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5821 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5881 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5941 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6001 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6061 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6121 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6181 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6241 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6301 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6361 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6421 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6481 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6541 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6661 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6721 ccctatggtg cactctcagt acaatctgct ctgatgccg
//
