LOCUS       sequence-307906-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp13 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  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 6981)
  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-Nsp13_GC3opt
FEATURES             Location/Qualifiers
     source          1..6981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        472..675
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     626..646
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    677..695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    698..716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             847..870
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             883..906
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2790..2807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2796..3020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3153..3172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3304..3351
                     /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             3359..4381
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3359..4381
                     /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    4511..4632
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4548..4567)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4602..4621
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4681..4697)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4681..4697)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4694..4716)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4705..4721
                     /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(4729..4735,4736..4753,4754..4759))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4774..4795
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4912..4929)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5083..5671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5163..5182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5842..6633,6634..6702))
                     /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     6465..6484
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6703..6807)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6882..6901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
       61 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      121 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      181 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      241 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      301 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      361 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      421 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      481 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      541 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      601 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      661 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
      721 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
      781 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt
      841 gagatggact ataaagatga cgatgacaag gccgcagccg cacatcacca ccatcatcac
      901 catcacggat ccgggatggc tgtgggagct tgcgtgctgt gtaatagcca gaccagcctg
      961 agatgcggcg cctgtatcag acggcctttc ctgtgctgca agtgctgcta cgaccacgtg
     1021 atcagcacca gccacaaact ggtgctgtcc gtgaatccct acgtgtgcaa tgcccctggc
     1081 tgcgacgtga ccgatgtgac acagctgtat ctcggcggca tgagctacta ctgcaagagc
     1141 cacaagcctc caatcagctt ccctctgtgc gccaacggac aggtgttcgg cctgtacaag
     1201 aatacctgcg tgggcagcga caatgtgacc gacttcaacg ccattgccac ctgtgactgg
     1261 accaacgccg gcgattacat cctggccaat acctgcaccg agcggctgaa gctgtttgcc
     1321 gccgaaacac tgaaggccac cgaggaaacc ttcaagctga gctacggaat cgccaccgtg
     1381 cgggaagtgc tgagcgatag agaactgcac ctgagctggg aagtgggcaa gcctagacct
     1441 cctctgaacc ggaactacgt gttcaccggc tacagagtga ccaagaacag caaggtgcag
     1501 atcggcgagt acaccttcga gaagggcgat tatggcgacg ccgtggtgta cagaggcacc
     1561 accacctaca agctgaacgt gggcgactac ttcgtgctga caagccacac cgtgatgcct
     1621 ctgtctgccc ctacactggt gccccaagaa cactacgtgc ggatcaccgg actgtacccc
     1681 acactgaaca tcagcgacga gttcagcagc aacgtggcca actaccagaa agtgggcatg
     1741 cagaagtaca gcacactgca gggcccacct ggcacaggca aaagccactt tgccattgga
     1801 ctggccctgt actaccccag cgccagaatc gtgtacaccg cctgttctca cgccgccgtt
     1861 gatgccctgt gtgaaaaggc cctgaagtac ctgcctatcg acaagtgcag ccggatcatc
     1921 cctgccagag ccagagtgga atgcttcgac aagttcaaag tgaacagcac cctggaacag
     1981 tacgtgttct gcaccgtgaa cgccctgcct gagacaacag ccgacatcgt ggtgttcgac
     2041 gagatctcca tggccaccaa ctacgacctg agcgtggtca acgccagact gagagccaag
     2101 cactacgtgt acatcggcga ccctgctcag ctgcccgctc ctagaacact gctgacaaag
     2161 ggcacactgg aacccgagta cttcaacagc gtgtgccggc tgatgaagac aatcggcccc
     2221 gacatgttcc tgggcacctg tagaagatgc cctgccgaga tcgtggacac cgtgtctgcc
     2281 ctggtgtacg acaacaaact gaaagcccac aaggacaaga gcgcccagtg cttcaagatg
     2341 ttctacaagg gcgtgatcac ccacgacgtg tccagcgcca tcaacagacc tcagatcgga
     2401 gttgtgcgcg agttcctgac cagaaatcct gcctggcgga aggccgtgtt tatcagcccc
     2461 tacaacagcc agaatgccgt ggccagcaag atcctgggac tgcctacaca gaccgtggat
     2521 agctctcagg gcagcgagta cgattacgtg atcttcaccc agaccaccga gacagcccac
     2581 agctgcaacg tgaacagatt caatgtggcc atcaccaggg ccaaagtggg aatcctgtgc
     2641 atcatgagcg accgggacct gtacgataag ctgcagttca ccagcctgga aatccccaga
     2701 cggaacgtgg caacactgca ggcctaggag atatccagca cagtggcggc cgctcgagtc
     2761 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2821 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2881 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2941 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3001 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3061 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3121 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3181 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3241 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     3301 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     3361 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     3421 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     3481 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     3541 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     3601 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     3661 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     3721 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     3781 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     3841 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3901 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3961 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     4021 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     4081 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     4141 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     4201 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     4261 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     4321 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     4381 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4441 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4501 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4561 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4621 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4681 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4741 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4801 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4861 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4921 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4981 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5041 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5101 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5161 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5221 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5281 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5341 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5401 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5461 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5521 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5581 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5641 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5701 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5761 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5821 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5881 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5941 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6001 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6061 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6121 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6181 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6241 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6301 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6361 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6421 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6481 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6541 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6601 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6661 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6721 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6781 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6841 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6901 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     6961 agcaaaattt aagctacaac a
//
