LOCUS       sequence-307837-        5718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged SARS-CoV-2 Nsp8 under control of a 
            tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5718)
  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 5718)
  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-Nsp8
FEATURES             Location/Qualifiers
     source          1..5718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        93..472
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        473..676
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     627..647
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    678..696
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    699..717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     727..751
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(1528..1545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1534..1758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1891..1910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2042..2089
                     /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             2097..3119
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2097..3119
                     /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    3249..3370
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3286..3305)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3340..3359
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3419..3435)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3419..3435)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3432..3454)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3443..3459
                     /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(3467..3473,3474..3491,3492..3497))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3512..3533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3650..3667)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3821..4409)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3901..3920)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4580..5371,5372..5440))
                     /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     5203..5222
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5441..5545)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5620..5639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
       61 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      121 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      241 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      301 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      361 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      421 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      481 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      541 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      601 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      661 ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg
      721 tcgacgagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
      781 cctccataga agacaccggg accgatccag cctccggact ctagcgttta aacttaagct
      841 tggtaccgag ctcggatccg ggatggctat cgcgtctgag ttctcctctc tgccaagtta
      901 cgctgcattt gcaacagcgc aggaagcata cgaacaggcc gttgcgaacg gcgacagtga
      961 agtcgtactg aaaaaactta aaaagtccct taatgttgcc aagtctgagt tcgaccgcga
     1021 cgcggccatg cagaggaaac tcgagaagat ggctgatcaa gccatgactc aaatgtataa
     1081 acaggcccgc tcagaggaca agagagccaa ggtaacgtct gctatgcaga cgatgctgtt
     1141 cacaatgctt agaaagcttg ataatgatgc gcttaataat atcatcaata atgctagaga
     1201 cgggtgcgtc ccgctcaata tcattcctct gacgacggcc gctaagttga tggtcgtgat
     1261 ccctgactac aacacttata agaacacctg cgacggaact acctttacct acgcgagtgc
     1321 gttgtgggaa atacagcaag tagtggatgc agacagcaag atagtacaac tgagcgagat
     1381 ttctatggac aactcaccta acctggcgtg gccactcatt gtcactgcgt tgcgggccaa
     1441 tagcgcggtt aaacttcagg cctaggagat atccagcaca gtggcggccg ctcgagtcta
     1501 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1561 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1621 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1681 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1741 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     1801 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1861 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1921 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1981 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     2041 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     2101 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     2161 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     2221 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     2281 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     2341 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     2401 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     2461 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     2521 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     2581 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     2641 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     2701 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     2761 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     2821 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     2881 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     2941 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     3001 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     3061 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     3121 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3181 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3241 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3301 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3361 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3421 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3481 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3541 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3601 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3661 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3721 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3781 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3841 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3901 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3961 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4021 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4081 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4141 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4201 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4261 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4321 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4381 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4441 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4501 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4561 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4621 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4681 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4741 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4801 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4861 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4921 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4981 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5041 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5101 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5161 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5221 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5281 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5341 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5401 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5461 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5521 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     5581 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     5641 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     5701 caaaatttaa gctacaac
//
