LOCUS       sequence-307915-        6749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag 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 6749)
  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 6749)
  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-Nsp14-HF
FEATURES             Location/Qualifiers
     source          1..6749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        135..514
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..718
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     669..689
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    720..738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    741..759
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     769..793
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2501..2524
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2537..2560
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2601..2618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2607..2831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2964..2983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3115..3162
                     /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             3170..4192
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3170..4192
                     /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    4322..4443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4359..4378)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4413..4432
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4492..4508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4492..4508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4505..4527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4516..4532
                     /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(4540..4546,4547..4564,4565..4570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4585..4606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4723..4740)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4894..5482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4974..4993)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5653..6444,6445..6513))
                     /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     6276..6295
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6514..6618)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6693..6712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
       61 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      121 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      181 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      241 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      301 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      361 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      421 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      481 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      541 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      601 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      661 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      721 ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt
      781 gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg
      841 ggaccgatcc agcctccgga ctctagcgtt taaacttaag cttggtaccg agctcggatc
      901 cgggatggct gaaaatgtga ctggtctctt caaagattgc tccaaggtga ttacgggcct
      961 gcatcccacc caggccccca cacatctttc agtagacacg aaatttaaaa ctgaaggtct
     1021 ttgtgttgac atccctggca ttccgaagga tatgacttat cgccggctta tctccatgat
     1081 ggggtttaag atgaattacc aggtcaacgg gtacccaaac atgttcatca cacgagagga
     1141 ggcaattcgg cacgttagag catggattgg ttttgacgtt gagggatgcc acgccactcg
     1201 agaggcggtc gggaccaact tgccactcca gttgggcttc tccaccggcg tcaatctggt
     1261 cgcagtaccc acaggttacg tggacacgcc caataacaca gacttttcaa gagtaagcgc
     1321 taaaccccca cccggagatc agttcaaaca cttgataccc ctgatgtaca aaggtcttcc
     1381 ctggaacgtt gtccgaatca aaatcgtcca gatgctctct gatacgctca agaacctgag
     1441 cgatcgagtc gttttcgtac tctgggcgca tggtttcgag ctgacctcca tgaagtactt
     1501 cgtaaaaata gggcctgaga gaacatgctg tctgtgtgat cgaagagcta cctgttttag
     1561 caccgcttcc gatacatatg cttgctggca tcattcaata ggtttcgatt acgtatacaa
     1621 cccattcatg atcgatgtgc agcagtgggg gttcactggg aacctgcaat caaaccacga
     1681 tctgtattgt caagtacacg gtaatgcgca cgtcgcttca tgtgatgcca tcatgacgcg
     1741 gtgcctggca gtacatgagt gctttgtaaa gagggttgat tggacgatag agtatcctat
     1801 tatcggagac gaactcaaaa taaatgctgc ctgccgcaaa gttcagcata tggttgtaaa
     1861 agcggccctt ctcgcggata aatttccggt tttgcatgac atcggaaatc cgaaagcaat
     1921 aaagtgcgtc ccgcaggcag acgtggaatg gaaattttat gatgcacaac cttgcagtga
     1981 taaggcctat aaaattgaag aactgttcta cagctacgcc acccactctg ataagttcac
     2041 agacggagtc tgtctttttt ggaattgtaa cgtcgatcgg tatccagcta attctattgt
     2101 ctgtcgattt gacacgaggg ttttgtcaaa cctgaacctt ccaggatgtg atggaggttc
     2161 tttgtacgtc aataaacacg cattccacac gcccgccttt gataaatctg cattcgtaaa
     2221 tctcaagcag cttcccttct tttactacag tgattcccca tgtgaatcac atggcaagca
     2281 agttgtcagc gatatagact atgtgcccct taagtctgca acctgcatca cgcgctgtaa
     2341 tttgggaggt gcagtctgtc ggcatcatgc gaatgagtat aggctttatc tcgacgcgta
     2401 caacatgatg atttcagcgg ggttttcatt gtgggtatat aagcaattcg acacttataa
     2461 cttgtggaac acatttacgc gcctccaggc atcgggcgga catcaccacc atcatcacca
     2521 tcacgccgca gccgcagatt ataaagatga cgatgacaag tagctcgagt ctagagggcc
     2581 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2641 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2701 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2761 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2821 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     2881 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2941 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3001 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3061 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt
     3121 cctattccga agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga
     3181 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     3241 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     3301 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     3361 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     3421 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     3481 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
     3541 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     3601 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     3661 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     3721 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     3781 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     3841 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     3901 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     3961 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     4021 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     4081 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     4141 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact
     4201 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4261 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4321 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4381 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4441 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4501 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4561 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4621 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4681 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4741 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4801 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4861 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4921 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4981 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5041 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5101 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5161 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5221 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5281 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5341 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5401 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5461 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5521 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5581 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5641 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5701 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5761 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5821 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5881 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5941 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6001 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6061 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6121 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6181 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6241 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6301 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6361 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6421 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6481 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6541 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6601 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     6661 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6721 agccagtatc tgctccctgc ttgtgtgtt
//
