LOCUS       sequence-307929-        6072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp16 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6072)
  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 6072)
  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-Nsp16
FEATURES             Location/Qualifiers
     source          1..6072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        136..515
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        516..719
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     670..690
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    721..739
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    742..760
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     770..794
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             891..914
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             927..950
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1925..1942)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1931..2155
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2288..2307)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2439..2486
                     /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             2494..3516
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2494..3516
                     /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    3646..3767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3683..3702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3737..3756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3816..3832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3816..3832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3829..3851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3840..3856
                     /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(3864..3870,3871..3888,3889..3894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3909..3930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4047..4064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4218..4806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4298..4317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4977..5768,5769..5837))
                     /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     5600..5619
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5838..5942)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6017..6036)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
       61 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
      121 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
      181 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      241 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      301 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      361 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      421 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      481 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      541 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      601 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      661 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
      721 tccctatcag tgatagagat ctccctatca gtgatagaga tcgtcgacga gctcgtttag
      781 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      841 gggaccgatc cagcctccgg actctagcgt ttaaacttaa gcttgagatg gactataaag
      901 atgacgatga caaggccgca gccgcacatc accaccatca tcaccatcac ggatccggga
      961 tgtcttctca agcctggcag cctggcgttg ctatgcccaa tttgtacaaa atgcagcgga
     1021 tgttgcttga aaaatgcgac cttcaaaact acggcgactc agccaccctc cctaagggaa
     1081 ttatgatgaa cgttgcgaag tacacacaac tgtgccagta cttgaacacc cttacccttg
     1141 ccgtcccata caacatgcgg gtcattcact ttggagcagg gagtgacaaa ggagtggcgc
     1201 cgggtactgc tgtgcttcga caatggttgc ccactggaac cctcctggtg gatagcgacc
     1261 tcaatgattt tgtttcagat gcagattcta ctttgattgg cgattgcgcg acagtacaca
     1321 ccgcgaacaa atgggacctt attatttcag acatgtacga tccaaagacc aaaaatgtca
     1381 ccaaggagaa tgactcaaag gagggctttt tcacgtacat atgcgggttt atacaacaaa
     1441 aactcgcctt gggaggtagc gtagctataa agattacgga gcacagttgg aatgcggatc
     1501 tgtataagct catgggccat tttgcatggt ggaccgcgtt tgttactaac gtcaacgcct
     1561 cttccagtga ggcatttctg attggatgca actatcttgg caaacccaga gaacagatcg
     1621 acggatacgt tatgcacgca aattatatat tttggcggaa taccaatccg atacaacttt
     1681 cctcctactc cttgtttgac atgtccaaat tccctctcaa gctgagaggc accgccgtta
     1741 tgagccttaa agagggtcag atcaacgata tgatcctttc ccttctttcc aaaggtcgcc
     1801 ttataataag ggagaacaat cgggttgtaa tttcaagtga cgttctggtc aataacgcct
     1861 aggagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     1921 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1981 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2041 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2101 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2161 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2221 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2281 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2341 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2401 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     2461 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     2521 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2581 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2641 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2701 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2761 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2821 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2881 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2941 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3001 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3061 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3121 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3181 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3241 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3301 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3361 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3421 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     3481 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3541 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3601 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3661 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3721 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3781 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3841 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3901 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3961 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4021 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4081 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4141 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4201 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4261 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4321 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4381 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4441 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4501 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4561 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4621 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4681 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4741 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4801 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4861 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4921 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4981 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5041 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5101 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5161 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5221 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5281 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5341 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5401 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5461 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5521 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5581 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5641 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5701 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5761 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5821 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5881 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5941 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     6001 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6061 ctgcttgtgt gt
//
