LOCUS       sequence-307913-        6705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged 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 6705)
  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 6705)
  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
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        151..530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        531..734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     685..705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    736..754
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    757..775
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     785..809
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2573..2590)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2579..2803
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2936..2955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3087..3134
                     /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             3142..4164
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3142..4164
                     /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    4294..4415
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4331..4350)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4385..4404
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4464..4480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4464..4480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4477..4499)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4488..4504
                     /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(4512..4518,4519..4536,4537..4542))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4557..4578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4695..4712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4866..5454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4946..4965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5625..6416,6417..6485))
                     /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     6248..6267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6486..6590)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6665..6684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
       61 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
      121 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      541 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      601 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      661 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      721 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      781 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      841 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttg
      901 gtaccgagct cggatccggg atggctgaaa atgtgactgg tctcttcaaa gattgctcca
      961 aggtgattac gggcctgcat cccacccagg cccccacaca tctttcagta gacacgaaat
     1021 ttaaaactga aggtctttgt gttgacatcc ctggcattcc gaaggatatg acttatcgcc
     1081 ggcttatctc catgatgggg tttaagatga attaccaggt caacgggtac ccaaacatgt
     1141 tcatcacacg agaggaggca attcggcacg ttagagcatg gattggtttt gacgttgagg
     1201 gatgccacgc cactcgagag gcggtcggga ccaacttgcc actccagttg ggcttctcca
     1261 ccggcgtcaa tctggtcgca gtacccacag gttacgtgga cacgcccaat aacacagact
     1321 tttcaagagt aagcgctaaa cccccacccg gagatcagtt caaacacttg atacccctga
     1381 tgtacaaagg tcttccctgg aacgttgtcc gaatcaaaat cgtccagatg ctctctgata
     1441 cgctcaagaa cctgagcgat cgagtcgttt tcgtactctg ggcgcatggt ttcgagctga
     1501 cctccatgaa gtacttcgta aaaatagggc ctgagagaac atgctgtctg tgtgatcgaa
     1561 gagctacctg ttttagcacc gcttccgata catatgcttg ctggcatcat tcaataggtt
     1621 tcgattacgt atacaaccca ttcatgatcg atgtgcagca gtgggggttc actgggaacc
     1681 tgcaatcaaa ccacgatctg tattgtcaag tacacggtaa tgcgcacgtc gcttcatgtg
     1741 atgccatcat gacgcggtgc ctggcagtac atgagtgctt tgtaaagagg gttgattgga
     1801 cgatagagta tcctattatc ggagacgaac tcaaaataaa tgctgcctgc cgcaaagttc
     1861 agcatatggt tgtaaaagcg gcccttctcg cggataaatt tccggttttg catgacatcg
     1921 gaaatccgaa agcaataaag tgcgtcccgc aggcagacgt ggaatggaaa ttttatgatg
     1981 cacaaccttg cagtgataag gcctataaaa ttgaagaact gttctacagc tacgccaccc
     2041 actctgataa gttcacagac ggagtctgtc ttttttggaa ttgtaacgtc gatcggtatc
     2101 cagctaattc tattgtctgt cgatttgaca cgagggtttt gtcaaacctg aaccttccag
     2161 gatgtgatgg aggttctttg tacgtcaata aacacgcatt ccacacgccc gcctttgata
     2221 aatctgcatt cgtaaatctc aagcagcttc ccttctttta ctacagtgat tccccatgtg
     2281 aatcacatgg caagcaagtt gtcagcgata tagactatgt gccccttaag tctgcaacct
     2341 gcatcacgcg ctgtaatttg ggaggtgcag tctgtcggca tcatgcgaat gagtataggc
     2401 tttatctcga cgcgtacaac atgatgattt cagcggggtt ttcattgtgg gtatataagc
     2461 aattcgacac ttataacttg tggaacacat ttacgcgcct ccaggcctag gagatatcca
     2521 gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     2581 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2641 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2701 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2761 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2821 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2881 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2941 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3001 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3061 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3121 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3181 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3241 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3301 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     3361 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     3421 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     3481 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     3541 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     3601 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     3661 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     3721 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     3781 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     3841 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     3901 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     3961 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4021 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4081 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4141 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4201 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4261 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4321 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4381 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4441 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4501 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4561 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4621 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4681 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4741 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4801 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4861 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4921 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4981 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5041 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5101 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5161 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5221 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5281 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5341 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5401 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5461 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5521 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5581 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5641 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5701 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5761 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5821 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5881 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5941 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6001 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6061 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6121 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6181 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6241 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6301 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6361 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6421 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6481 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6541 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6601 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     6661 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgc
//
