LOCUS       sequence-307912-        6971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag 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 6971)
  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 6971)
  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-Nsp13-HF_GC3opt
FEATURES             Location/Qualifiers
     source          1..6971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        132..511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        512..715
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     666..686
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    717..735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    738..756
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     766..790
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2720..2743
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2756..2779
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2820..2837)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2826..3050
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3183..3202)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3334..3381
                     /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             3389..4411
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3389..4411
                     /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    4541..4662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4578..4597)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4632..4651
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4711..4727)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4711..4727)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4724..4746)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4735..4751
                     /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(4759..4765,4766..4783,4784..4789))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4804..4825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4942..4959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5113..5701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5193..5212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5872..6663,6664..6732))
                     /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     6495..6514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6733..6837)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6912..6931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
       61 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      121 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      181 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      241 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      301 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      361 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      421 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      481 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      541 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      601 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      661 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc
      721 tatcagtgat agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa
      781 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga
      841 ccgatccagc ctccggactc tagcgtttaa acttaagctt ggtaccgagc tcggatccgg
      901 gatggctgtg ggagcttgcg tgctgtgtaa tagccagacc agcctgagat gcggcgcctg
      961 tatcagacgg cctttcctgt gctgcaagtg ctgctacgac cacgtgatca gcaccagcca
     1021 caaactggtg ctgtccgtga atccctacgt gtgcaatgcc cctggctgcg acgtgaccga
     1081 tgtgacacag ctgtatctcg gcggcatgag ctactactgc aagagccaca agcctccaat
     1141 cagcttccct ctgtgcgcca acggacaggt gttcggcctg tacaagaata cctgcgtggg
     1201 cagcgacaat gtgaccgact tcaacgccat tgccacctgt gactggacca acgccggcga
     1261 ttacatcctg gccaatacct gcaccgagcg gctgaagctg tttgccgccg aaacactgaa
     1321 ggccaccgag gaaaccttca agctgagcta cggaatcgcc accgtgcggg aagtgctgag
     1381 cgatagagaa ctgcacctga gctgggaagt gggcaagcct agacctcctc tgaaccggaa
     1441 ctacgtgttc accggctaca gagtgaccaa gaacagcaag gtgcagatcg gcgagtacac
     1501 cttcgagaag ggcgattatg gcgacgccgt ggtgtacaga ggcaccacca cctacaagct
     1561 gaacgtgggc gactacttcg tgctgacaag ccacaccgtg atgcctctgt ctgcccctac
     1621 actggtgccc caagaacact acgtgcggat caccggactg taccccacac tgaacatcag
     1681 cgacgagttc agcagcaacg tggccaacta ccagaaagtg ggcatgcaga agtacagcac
     1741 actgcagggc ccacctggca caggcaaaag ccactttgcc attggactgg ccctgtacta
     1801 ccccagcgcc agaatcgtgt acaccgcctg ttctcacgcc gccgttgatg ccctgtgtga
     1861 aaaggccctg aagtacctgc ctatcgacaa gtgcagccgg atcatccctg ccagagccag
     1921 agtggaatgc ttcgacaagt tcaaagtgaa cagcaccctg gaacagtacg tgttctgcac
     1981 cgtgaacgcc ctgcctgaga caacagccga catcgtggtg ttcgacgaga tctccatggc
     2041 caccaactac gacctgagcg tggtcaacgc cagactgaga gccaagcact acgtgtacat
     2101 cggcgaccct gctcagctgc ccgctcctag aacactgctg acaaagggca cactggaacc
     2161 cgagtacttc aacagcgtgt gccggctgat gaagacaatc ggccccgaca tgttcctggg
     2221 cacctgtaga agatgccctg ccgagatcgt ggacaccgtg tctgccctgg tgtacgacaa
     2281 caaactgaaa gcccacaagg acaagagcgc ccagtgcttc aagatgttct acaagggcgt
     2341 gatcacccac gacgtgtcca gcgccatcaa cagacctcag atcggagttg tgcgcgagtt
     2401 cctgaccaga aatcctgcct ggcggaaggc cgtgtttatc agcccctaca acagccagaa
     2461 tgccgtggcc agcaagatcc tgggactgcc tacacagacc gtggatagct ctcagggcag
     2521 cgagtacgat tacgtgatct tcacccagac caccgagaca gcccacagct gcaacgtgaa
     2581 cagattcaat gtggccatca ccagggccaa agtgggaatc ctgtgcatca tgagcgaccg
     2641 ggacctgtac gataagctgc agttcaccag cctggaaatc cccagacgga acgtggcaac
     2701 actgcaggca tcgggcggac atcaccacca tcatcaccat cacgccgcag ccgcagatta
     2761 taaagatgac gatgacaagt agctcgagtc tagagggccc gtttaaaccc gctgatcagc
     2821 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2881 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2941 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3001 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3061 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3121 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3181 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3241 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3301 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     3361 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     3421 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     3481 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     3541 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     3601 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     3661 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     3721 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     3781 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     3841 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     3901 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     3961 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     4021 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     4081 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     4141 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     4201 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     4261 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     4321 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     4381 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     4441 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4501 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4561 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4621 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4681 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4741 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4801 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4861 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4921 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4981 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5041 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5101 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5161 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5221 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5281 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5341 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5401 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5461 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5521 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5581 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5641 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5701 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5761 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6721 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6781 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6841 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     6901 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     6961 tgtgtgttgg a
//
