LOCUS       sequence-307867-        5463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged SARS-CoV-2 Nsp9 under control of a 
            tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5463)
  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 5463)
  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-Nsp9
FEATURES             Location/Qualifiers
     source          1..5463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        107..486
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        487..690
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     641..661
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    692..710
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    713..731
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     741..765
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(1287..1304)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1293..1517
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1650..1669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1801..1848
                     /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             1856..2878
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1856..2878
                     /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    3008..3129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3045..3064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3099..3118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3178..3194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3178..3194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3191..3213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3202..3218
                     /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(3226..3232,3233..3250,3251..3256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3271..3292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3409..3426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3580..4168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3660..3679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4339..5130,5131..5199))
                     /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     4962..4981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5200..5304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5379..5398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
       61 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      121 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      181 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      241 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      301 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      361 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      421 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      481 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      541 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      601 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      661 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
      721 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
      781 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg
      841 tttaaactta agcttggtac cgagctcgga tccgggatga ataatgaact tagccccgtt
      901 gcgttgagac aaatgtcttg cgccgccgga acgactcaga cggcctgtac tgatgacaat
      961 gctcttgcat attataatac tacaaagggc ggtcggtttg tgttggctct tctttccgat
     1021 ctgcaagatt tgaaatgggc gcggtttccg aaaagcgatg gaacaggcac catttacacg
     1081 gaattggaac cgccttgccg attcgtgacg gatactccta aggggcctaa agtgaaatac
     1141 ttgtacttta tcaagggtct taacaatttg aaccggggta tggtgctcgg cagtctggct
     1201 gcaacggtcc gcctgcaagc ctaggagata tccagcacag tggcggccgc tcgagtctag
     1261 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1321 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1381 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1441 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1501 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     1561 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1621 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1681 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1741 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta
     1801 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa
     1861 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     1921 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     1981 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     2041 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     2101 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     2161 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     2221 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     2281 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     2341 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     2401 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     2461 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     2521 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     2581 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     2641 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     2701 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     2761 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     2821 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca
     2881 cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     2941 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     3001 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3061 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3121 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     3181 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3241 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3421 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3481 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4441 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     5341 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     5401 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     5461 aaa
//
