LOCUS       sequence-307870-        5507 bp    DNA     circular SYN 14-APR-2025
DEFINITION  mammalian expression of C-terminally His8-Flag 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 5507)
  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 5507)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-HF
FEATURES             Location/Qualifiers
     source          1..5507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        507..710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     661..681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    712..730
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    733..751
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     761..785
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1251..1274
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1287..1310
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1351..1368)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1357..1581
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1714..1733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1865..1912
                     /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             1920..2942
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1920..2942
                     /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    3072..3193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3109..3128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3163..3182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3242..3258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3242..3258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3255..3277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3266..3282
                     /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(3290..3296,3297..3314,3315..3320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3335..3356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3473..3490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3644..4232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3724..3743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4403..5194,5195..5263))
                     /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     5026..5045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5264..5368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5443..5462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
       61 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      121 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
      721 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
      781 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      841 ccagcctccg gactctagcg tttaaactta agcttggtac cgagctcgga tccgggatga
      901 ataatgaact tagccccgtt gcgttgagac aaatgtcttg cgccgccgga acgactcaga
      961 cggcctgtac tgatgacaat gctcttgcat attataatac tacaaagggc ggtcggtttg
     1021 tgttggctct tctttccgat ctgcaagatt tgaaatgggc gcggtttccg aaaagcgatg
     1081 gaacaggcac catttacacg gaattggaac cgccttgccg attcgtgacg gatactccta
     1141 aggggcctaa agtgaaatac ttgtacttta tcaagggtct taacaatttg aaccggggta
     1201 tggtgctcgg cagtctggct gcaacggtcc gcctgcaagc atcgggcgga catcaccacc
     1261 atcatcacca tcacgccgca gccgcagatt ataaagatga cgatgacaag tagctcgagt
     1321 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1381 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1441 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1501 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1561 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     1621 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1681 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1741 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1801 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1861 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     1921 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     1981 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     2041 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     2101 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     2161 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     2221 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     2281 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     2341 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     2401 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     2461 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     2521 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     2581 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     2641 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     2701 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     2761 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     2821 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     2881 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     2941 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3001 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3061 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3121 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3181 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3241 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3301 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3361 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3421 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3481 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3541 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3601 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3661 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3721 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3781 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3841 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3901 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3961 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4021 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4081 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4141 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4201 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4261 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4321 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4381 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4441 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4501 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4561 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4621 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4681 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4741 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4801 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4861 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4921 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4981 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5041 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5101 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5161 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5221 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5281 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5341 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     5401 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     5461 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcg
//
