LOCUS       sequence-307902-        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
FEATURES             Location/Qualifiers
     source          1..6927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        185..564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        565..768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     719..739
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    770..788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    791..809
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     819..843
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2829..2846)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2835..3059
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3192..3211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3343..3390
                     /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             3398..4420
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3398..4420
                     /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    4550..4671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4587..4606)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4641..4660
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4720..4736)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4720..4736)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4733..4755)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4744..4760
                     /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(4768..4774,4775..4792,4793..4798))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4813..4834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4951..4968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5122..5710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5202..5221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5881..6672,6673..6741))
                     /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     6504..6523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6742..6846)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6921..6927,1..13))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
       61 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      121 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      181 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt
      781 gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag
      841 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
      901 agcctccgga ctctagcgtt taaacttaag cttggtaccg agctcggatc cgggatggct
      961 gttggagcct gtgtgttgtg taacagccag acttccctga gatgcggggc gtgcatacgc
     1021 aggccgtttc tgtgctgtaa gtgttgttac gatcacgtca tttctacaag ccacaagctg
     1081 gtcttgagcg ttaaccctta tgtttgtaat gcccctggct gcgatgttac tgatgtcacc
     1141 cagttgtatt tgggtgggat gtcatattac tgtaagagcc ataagccacc gatctcattc
     1201 cccttgtgcg cgaacggaca agtatttggt ttgtacaaaa acacgtgtgt aggatcagat
     1261 aacgtaaccg atttcaatgc tattgcgacg tgtgattgga caaatgcagg agattacatt
     1321 cttgccaata cttgcacaga aagacttaaa ttgttcgctg cggaaacatt gaaagcaact
     1381 gaagagacgt ttaagctgtc atacggcatt gccaccgtga gggaggtgct gtctgaccga
     1441 gagctccatc tgtcctggga ggtcgggaaa ccccgacctc cgttgaacag aaactacgtg
     1501 tttaccggtt accgcgtcac aaagaattca aaggtgcaga taggagaata tactttcgaa
     1561 aagggagatt acggagacgc ggtggtttat cgaggcacga cgacctacaa gctgaatgta
     1621 ggcgattatt ttgtccttac atctcatacg gtcatgccat tgagcgctcc cactctggtc
     1681 ccacaggaac attatgtgag gattacaggc ctgtatccca ctttgaacat aagtgatgaa
     1741 ttttcctcta atgtcgcaaa ttaccaaaaa gtaggtatgc agaagtattc cactctccag
     1801 gggccgcccg gaactgggaa gagtcacttc gcaattgggt tggctctgta ttatccaagc
     1861 gcacgaatag tttacacagc atgtagccat gcggccgttg atgcgctgtg cgagaaagcg
     1921 ctgaaatacc ttcctattga caaatgcagc cggatcatcc ccgcacgcgc ccgcgtggag
     1981 tgttttgata agttcaaagt aaattcaacc ctggagcagt atgtgttttg tacggtgaat
     2041 gccctgccag aaacaactgc ggacatagtg gtgtttgacg aaatatcaat ggcgactaat
     2101 tatgacctta gtgtggtgaa cgctagattg cgagccaagc actacgtata tataggcgat
     2161 ccagctcaac tccccgctcc tcggacgctc ctgaccaagg gaaccttgga gccagaatac
     2221 ttcaattccg tctgccgact gatgaagacc ataggcccag acatgttctt ggggacgtgt
     2281 cgccgctgtc cagcagaaat agtggacacg gttagcgcgc ttgtgtacga caataaactc
     2341 aaggcacaca aagataagtc cgcccagtgt tttaaaatgt tctataaagg tgttataact
     2401 catgatgtct cctctgcaat caatcgaccc cagatcgggg ttgtccgaga gtttctgaca
     2461 cggaatcccg cttggcgaaa ggcggtgttt atctctccat acaactctca gaatgccgtg
     2521 gcctctaaga tccttggtct gcccacgcaa accgtagatt ctagccaggg tagcgagtat
     2581 gactacgtta tttttactca gacaacagaa actgctcata gttgcaacgt caataggttc
     2641 aatgtggcca ttacaagggc gaaggtgggt attctgtgta ttatgagtga tcgagacctg
     2701 tacgataaac ttcagtttac ctcactggag atcccccgca gaaatgtggc caccctgcaa
     2761 gcctaggaga tatccagcac agtggcggcc gctcgagtct agagggcccg tttaaacccg
     2821 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2881 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2941 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3001 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3061 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     3361 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     3421 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     3481 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     3541 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3601 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3661 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3721 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3781 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3841 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3901 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3961 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     4021 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     4081 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     4141 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     4201 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     4261 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     4321 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     4381 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     4441 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4501 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4561 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4621 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4681 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4741 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4801 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4861 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4921 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4981 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5041 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5101 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5161 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5221 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5281 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5341 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5401 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5461 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5521 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5581 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5641 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5701 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5761 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5821 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5881 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5941 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6001 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6061 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6121 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6181 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6241 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6301 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6361 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6421 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6481 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6541 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6601 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6661 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6721 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6781 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6841 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     6901 cccctatggt gcactctcag tacaatc
//
