LOCUS       sequence-307903-        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
FEATURES             Location/Qualifiers
     source          1..6981
                     /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"
     CDS             906..929
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             942..965
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2849..2866)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2855..3079
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3212..3231)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3363..3410
                     /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             3418..4440
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3418..4440
                     /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    4570..4691
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4607..4626)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4661..4680
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4740..4756)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4740..4756)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4753..4775)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4764..4780
                     /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(4788..4794,4795..4812,4813..4818))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4833..4854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4971..4988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5142..5730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5222..5241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5901..6692,6693..6761))
                     /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     6524..6543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6762..6866)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6941..6960)
                     /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 agatggacta taaagatgac gatgacaagg ccgcagccgc acatcaccac catcatcacc
      961 atcacggatc cgggatggct gttggagcct gtgtgttgtg taacagccag acttccctga
     1021 gatgcggggc gtgcatacgc aggccgtttc tgtgctgtaa gtgttgttac gatcacgtca
     1081 tttctacaag ccacaagctg gtcttgagcg ttaaccctta tgtttgtaat gcccctggct
     1141 gcgatgttac tgatgtcacc cagttgtatt tgggtgggat gtcatattac tgtaagagcc
     1201 ataagccacc gatctcattc cccttgtgcg cgaacggaca agtatttggt ttgtacaaaa
     1261 acacgtgtgt aggatcagat aacgtaaccg atttcaatgc tattgcgacg tgtgattgga
     1321 caaatgcagg agattacatt cttgccaata cttgcacaga aagacttaaa ttgttcgctg
     1381 cggaaacatt gaaagcaact gaagagacgt ttaagctgtc atacggcatt gccaccgtga
     1441 gggaggtgct gtctgaccga gagctccatc tgtcctggga ggtcgggaaa ccccgacctc
     1501 cgttgaacag aaactacgtg tttaccggtt accgcgtcac aaagaattca aaggtgcaga
     1561 taggagaata tactttcgaa aagggagatt acggagacgc ggtggtttat cgaggcacga
     1621 cgacctacaa gctgaatgta ggcgattatt ttgtccttac atctcatacg gtcatgccat
     1681 tgagcgctcc cactctggtc ccacaggaac attatgtgag gattacaggc ctgtatccca
     1741 ctttgaacat aagtgatgaa ttttcctcta atgtcgcaaa ttaccaaaaa gtaggtatgc
     1801 agaagtattc cactctccag gggccgcccg gaactgggaa gagtcacttc gcaattgggt
     1861 tggctctgta ttatccaagc gcacgaatag tttacacagc atgtagccat gcggccgttg
     1921 atgcgctgtg cgagaaagcg ctgaaatacc ttcctattga caaatgcagc cggatcatcc
     1981 ccgcacgcgc ccgcgtggag tgttttgata agttcaaagt aaattcaacc ctggagcagt
     2041 atgtgttttg tacggtgaat gccctgccag aaacaactgc ggacatagtg gtgtttgacg
     2101 aaatatcaat ggcgactaat tatgacctta gtgtggtgaa cgctagattg cgagccaagc
     2161 actacgtata tataggcgat ccagctcaac tccccgctcc tcggacgctc ctgaccaagg
     2221 gaaccttgga gccagaatac ttcaattccg tctgccgact gatgaagacc ataggcccag
     2281 acatgttctt ggggacgtgt cgccgctgtc cagcagaaat agtggacacg gttagcgcgc
     2341 ttgtgtacga caataaactc aaggcacaca aagataagtc cgcccagtgt tttaaaatgt
     2401 tctataaagg tgttataact catgatgtct cctctgcaat caatcgaccc cagatcgggg
     2461 ttgtccgaga gtttctgaca cggaatcccg cttggcgaaa ggcggtgttt atctctccat
     2521 acaactctca gaatgccgtg gcctctaaga tccttggtct gcccacgcaa accgtagatt
     2581 ctagccaggg tagcgagtat gactacgtta tttttactca gacaacagaa actgctcata
     2641 gttgcaacgt caataggttc aatgtggcca ttacaagggc gaaggtgggt attctgtgta
     2701 ttatgagtga tcgagacctg tacgataaac ttcagtttac ctcactggag atcccccgca
     2761 gaaatgtggc caccctgcaa gcctaggaga tatccagcac agtggcggcc gctcgagtct
     2821 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     2881 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2941 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3001 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3061 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     3121 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3181 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3241 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3301 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     3361 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     3421 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     3481 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3541 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     3601 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     3661 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     3721 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     3781 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     3841 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     3901 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     3961 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4021 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4081 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4141 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4201 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4261 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4321 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4381 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     4441 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4501 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4561 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4621 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4681 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4741 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4801 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4861 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4921 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4981 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5041 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5101 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5161 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5221 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5281 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5341 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5401 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5461 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5521 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5581 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5641 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5701 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5761 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5821 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5881 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5941 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6001 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6061 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6121 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6181 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6241 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6301 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6361 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6421 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6481 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6541 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6601 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6661 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6721 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6781 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6841 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     6901 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     6961 catagttaag ccagtatctg c
//
