LOCUS       sequence-307872-        5595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp10 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5595)
  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 5595)
  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-Nsp10
FEATURES             Location/Qualifiers
     source          1..5595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..475
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        476..679
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     630..650
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    681..699
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    702..720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     730..754
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             851..874
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             887..910
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1408..1425)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1414..1638
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1771..1790)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1922..1969
                     /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             1977..2999
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1977..2999
                     /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    3129..3250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3166..3185)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3220..3239
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3299..3315)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3299..3315)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3312..3334)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3323..3339
                     /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(3347..3353,3354..3371,3372..3377))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3392..3413
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3530..3547)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3701..4289)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3781..3800)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4460..5251,5252..5320))
                     /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     5083..5102
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5321..5425)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5500..5519)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
       61 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      421 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      481 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      601 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      661 ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga
      721 tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      781 tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa
      841 gcttgagatg gactataaag atgacgatga caaggccgca gccgcacatc accaccatca
      901 tcaccatcac ggatccggga tggctggcaa cgcaacggag gttcctgcaa atagtacagt
      961 tctcagcttt tgcgcttttg cagttgacgc cgctaaggca tataaggatt atttggcatc
     1021 cgggggtcaa ccaatcacca actgcgtcaa aatgctctgc actcatacag gaaccggaca
     1081 ggcaatcact gtgactcctg aggcaaacat ggatcaggag agcttcggag gggccagttg
     1141 ctgtttgtac tgtagatgcc atatagatca tcctaacccc aaagggttct gcgatcttaa
     1201 aggtaaatat gtacaaatac ccacaacttg cgcgaacgac cccgttggtt ttacccttaa
     1261 aaatactgta tgcaccgtct gcgggatgtg gaagggttac ggctgctcat gtgaccaact
     1321 cagagagccc atgctgcagg cctaggagat atccagcaca gtggcggccg ctcgagtcta
     1381 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1441 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1501 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1561 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1621 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     1681 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1741 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1801 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1861 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     1921 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     1981 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     2041 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     2101 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     2161 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     2221 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     2281 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     2341 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     2401 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     2461 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     2521 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     2581 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     2641 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     2701 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     2761 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     2821 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     2881 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     2941 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     3001 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3061 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3121 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3181 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3241 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3301 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3361 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3421 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3481 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3541 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3601 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3661 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3721 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3781 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3841 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3901 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3961 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4021 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4081 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4141 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4201 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4261 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4321 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4381 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4441 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4501 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4561 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4621 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4681 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4741 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4801 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4861 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4921 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4981 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5041 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5101 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5161 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5221 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5281 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5341 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5401 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     5461 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     5521 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     5581 caaaatttaa gctac
//
