LOCUS       sequence-307834-        6057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged Nsp7-Nsp8 
            fusion protein under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6057)
  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 6057)
  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: pcDNA-FRT-TO-FH-Nsp7-Nsp8_GC3opt
FEATURES             Location/Qualifiers
     source          1..6057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..465
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        466..669
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     620..640
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    671..689
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    692..710
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     720..744
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             841..864
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             877..900
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1860..1877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1866..2090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2223..2242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2374..2421
                     /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             2429..3451
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2429..3451
                     /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    3581..3702
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3618..3637)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3672..3691
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3751..3767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3751..3767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3764..3786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3775..3791
                     /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(3799..3805,3806..3823,3824..3829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3844..3865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3982..3999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4153..4741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4233..4252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4912..5703,5704..5772))
                     /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     5535..5554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5773..5877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5952..5971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
       61 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      121 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      181 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      241 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      301 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      361 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      421 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      481 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      541 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      601 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      661 agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga tcgtcgacga
      721 gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat
      781 agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa gcttgagatg
      841 gactataaag atgacgatga caaggccgca gccgcacatc accaccatca tcaccatcac
      901 ggatccggga tgagcaagat gagcgacgtg aagtgcacca gcgtggtgct gctgtctgtt
      961 ctgcagcagc tgagagtgga aagcagcagc aaactgtggg cccagtgtgt ccagctgcac
     1021 aacgatattc tgctggccaa ggacaccacc gaggccttcg agaagatggt gtctctgctg
     1081 agcgtgctgc tgagcatgca aggcgccgtg gacatcaaca agctgtgcga ggaaatgctg
     1141 gacaaccggg ccacactgca gggctctgct ggatctgctg ctggctctgg cgaatttgcc
     1201 attgccagcg agttcagcag cctgcctagc tatgccgcct ttgccacagc tcaagaggcc
     1261 tatgaacagg ccgtggccaa tggcgattct gaggtggtgc tgaagaagct gaaaaagtcc
     1321 ctgaacgtgg ccaagagcga gttcgacaga gatgccgcca tgcagcggaa gctggaaaag
     1381 atggccgacc aggccatgac acagatgtac aagcaggcca gaagcgagga caagagggcc
     1441 aaagtgacct ctgccatgca gaccatgctg ttcaccatgc tgagaaagct ggataacgac
     1501 gccctgaaca acatcatcaa caacgccaga gatggctgcg tgcccctgaa tatcatccct
     1561 ctgacaaccg ccgccaagct gatggtggtc atccccgact acaacaccta caagaatacc
     1621 tgcgacggca ccaccttcac ctacgcctct gctctgtggg agattcagca ggtcgtggac
     1681 gccgacagca agatcgtgca gctgagcgag atcagcatgg acaacagccc caatctggcc
     1741 tggcctctga ttgtgacagc cctgagagcc aacagcgccg tgaaactgca ggcctaggag
     1801 atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc
     1861 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1921 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1981 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2041 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2101 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2161 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2221 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2281 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2341 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     2401 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     2461 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     2521 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     2581 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     2641 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     2701 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     2761 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     2821 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     2881 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     2941 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     3001 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     3061 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     3121 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     3181 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     3241 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     3301 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     3361 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     3421 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     3481 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3541 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3601 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3661 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3721 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3781 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3841 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3901 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3961 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4021 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4081 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4141 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4201 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4261 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4321 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4381 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4441 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4501 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4561 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4621 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4681 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4741 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4801 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4861 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4921 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4981 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5041 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5101 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5161 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5221 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5281 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5341 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5401 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5461 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5521 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5581 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5641 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5701 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5761 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5821 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5881 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     5941 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     6001 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaa
//
