LOCUS       sequence-307868-        5517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged 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 5517)
  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 5517)
  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-Nsp9
FEATURES             Location/Qualifiers
     source          1..5517
                     /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(1385..1402)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1391..1615
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1748..1767)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1899..1946
                     /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             1954..2976
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1954..2976
                     /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    3106..3227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3143..3162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3197..3216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3276..3292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3276..3292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3289..3311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3300..3316
                     /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(3324..3330,3331..3348,3349..3354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3369..3390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3507..3524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3678..4266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3758..3777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4437..5228,5229..5297))
                     /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     5060..5079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5298..5402)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5477..5496)
                     /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 cgggatgaat aatgaactta gccccgttgc gttgagacaa atgtcttgcg
     1021 ccgccggaac gactcagacg gcctgtactg atgacaatgc tcttgcatat tataatacta
     1081 caaagggcgg tcggtttgtg ttggctcttc tttccgatct gcaagatttg aaatgggcgc
     1141 ggtttccgaa aagcgatgga acaggcacca tttacacgga attggaaccg ccttgccgat
     1201 tcgtgacgga tactcctaag gggcctaaag tgaaatactt gtactttatc aagggtctta
     1261 acaatttgaa ccggggtatg gtgctcggca gtctggctgc aacggtccgc ctgcaagcct
     1321 aggagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     1381 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1441 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1501 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1561 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     1621 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     1681 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1741 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1801 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1861 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     1921 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     1981 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2041 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2101 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2161 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2221 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2281 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2341 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2401 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2461 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     2521 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     2581 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     2641 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     2701 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     2761 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     2821 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     2881 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     2941 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3001 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3061 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3121 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3181 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3241 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3301 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3361 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3421 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3481 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3541 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3601 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3661 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3721 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3781 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3841 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3901 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3961 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4021 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4081 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4141 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4201 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4261 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4321 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4381 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4441 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4501 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4561 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4621 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4681 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4741 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4801 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4861 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4921 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4981 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5041 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5101 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5161 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5221 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5281 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5341 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5401 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5461 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgc
//
