LOCUS       sequence-307905-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged 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 6927)
  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 6927)
  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_GC3opt
FEATURES             Location/Qualifiers
     source          1..6927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        117..496
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        497..700
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     651..671
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    702..720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    723..741
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     751..775
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2761..2778)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2767..2991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3124..3143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3275..3322
                     /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             3330..4352
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3330..4352
                     /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    4482..4603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4519..4538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4573..4592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4652..4668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4652..4668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4665..4687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4676..4692
                     /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(4700..4706,4707..4724,4725..4730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4745..4766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4883..4900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5054..5642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5134..5153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5813..6604,6605..6673))
                     /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     6436..6455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6674..6778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6853..6872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
       61 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      121 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      181 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      241 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      301 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      361 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      421 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      481 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      541 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      601 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      661 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga
      721 tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg
      781 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
      841 gactctagcg tttaaactta agcttggtac cgagctcgga tccgggatgg ctgtgggagc
      901 ttgcgtgctg tgtaatagcc agaccagcct gagatgcggc gcctgtatca gacggccttt
      961 cctgtgctgc aagtgctgct acgaccacgt gatcagcacc agccacaaac tggtgctgtc
     1021 cgtgaatccc tacgtgtgca atgcccctgg ctgcgacgtg accgatgtga cacagctgta
     1081 tctcggcggc atgagctact actgcaagag ccacaagcct ccaatcagct tccctctgtg
     1141 cgccaacgga caggtgttcg gcctgtacaa gaatacctgc gtgggcagcg acaatgtgac
     1201 cgacttcaac gccattgcca cctgtgactg gaccaacgcc ggcgattaca tcctggccaa
     1261 tacctgcacc gagcggctga agctgtttgc cgccgaaaca ctgaaggcca ccgaggaaac
     1321 cttcaagctg agctacggaa tcgccaccgt gcgggaagtg ctgagcgata gagaactgca
     1381 cctgagctgg gaagtgggca agcctagacc tcctctgaac cggaactacg tgttcaccgg
     1441 ctacagagtg accaagaaca gcaaggtgca gatcggcgag tacaccttcg agaagggcga
     1501 ttatggcgac gccgtggtgt acagaggcac caccacctac aagctgaacg tgggcgacta
     1561 cttcgtgctg acaagccaca ccgtgatgcc tctgtctgcc cctacactgg tgccccaaga
     1621 acactacgtg cggatcaccg gactgtaccc cacactgaac atcagcgacg agttcagcag
     1681 caacgtggcc aactaccaga aagtgggcat gcagaagtac agcacactgc agggcccacc
     1741 tggcacaggc aaaagccact ttgccattgg actggccctg tactacccca gcgccagaat
     1801 cgtgtacacc gcctgttctc acgccgccgt tgatgccctg tgtgaaaagg ccctgaagta
     1861 cctgcctatc gacaagtgca gccggatcat ccctgccaga gccagagtgg aatgcttcga
     1921 caagttcaaa gtgaacagca ccctggaaca gtacgtgttc tgcaccgtga acgccctgcc
     1981 tgagacaaca gccgacatcg tggtgttcga cgagatctcc atggccacca actacgacct
     2041 gagcgtggtc aacgccagac tgagagccaa gcactacgtg tacatcggcg accctgctca
     2101 gctgcccgct cctagaacac tgctgacaaa gggcacactg gaacccgagt acttcaacag
     2161 cgtgtgccgg ctgatgaaga caatcggccc cgacatgttc ctgggcacct gtagaagatg
     2221 ccctgccgag atcgtggaca ccgtgtctgc cctggtgtac gacaacaaac tgaaagccca
     2281 caaggacaag agcgcccagt gcttcaagat gttctacaag ggcgtgatca cccacgacgt
     2341 gtccagcgcc atcaacagac ctcagatcgg agttgtgcgc gagttcctga ccagaaatcc
     2401 tgcctggcgg aaggccgtgt ttatcagccc ctacaacagc cagaatgccg tggccagcaa
     2461 gatcctggga ctgcctacac agaccgtgga tagctctcag ggcagcgagt acgattacgt
     2521 gatcttcacc cagaccaccg agacagccca cagctgcaac gtgaacagat tcaatgtggc
     2581 catcaccagg gccaaagtgg gaatcctgtg catcatgagc gaccgggacc tgtacgataa
     2641 gctgcagttc accagcctgg aaatccccag acggaacgtg gcaacactgc aggcctagga
     2701 gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag
     2761 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2821 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2881 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2941 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3001 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3061 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3241 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     3301 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     3361 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     3421 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     3481 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     3541 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     3601 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     3661 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     3721 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     3781 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     3841 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     3901 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     3961 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4021 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4081 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4141 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4201 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4261 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4321 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     4381 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4441 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4501 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4561 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4621 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4681 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4741 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4801 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4861 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4921 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4981 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5041 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5101 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5161 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5221 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5281 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5341 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5401 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5461 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5521 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5581 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5641 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5701 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5761 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5821 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5881 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5941 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6001 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6061 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6121 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6181 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6241 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6301 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6361 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6421 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6481 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6541 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6601 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6661 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6721 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6781 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6901 ttgtgtgttg gaggtcgctg agtagtg
//
