LOCUS       sequence-307862-        5772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged 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 5772)
  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 5772)
  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-Nsp8_GC3opt
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        126..505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        506..709
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     660..680
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    711..729
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    732..750
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     760..784
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             881..904
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             917..940
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1615..1632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1621..1845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1978..1997)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2129..2176
                     /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             2184..3206
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2184..3206
                     /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    3336..3457
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3373..3392)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3427..3446
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3506..3522)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3506..3522)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3519..3541)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3530..3546
                     /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(3554..3560,3561..3578,3579..3584))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3599..3620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3737..3754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3908..4496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3988..4007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4667..5458,5459..5527))
                     /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     5290..5309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5528..5632)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5707..5726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
       61 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
      121 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      181 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      241 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      301 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      361 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      421 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      481 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      541 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      601 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      661 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tccctatcag
      721 tgatagagat ctccctatca gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca
      781 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
      841 cagcctccgg actctagcgt ttaaacttaa gcttgagatg gactataaag atgacgatga
      901 caaggccgca gccgcacatc accaccatca tcaccatcac ggatccggga tggccattgc
      961 cagcgagttt agcagcctgc ctagctacgc cgcctttgcc acagctcaag aggcctatga
     1021 acaggccgtg gccaatggcg attctgaggt ggtgctgaag aagctgaaaa agtccctgaa
     1081 cgtggccaag agcgagttcg acagagatgc cgccatgcag cggaagctgg aaaagatggc
     1141 cgaccaggcc atgacacaga tgtacaagca ggccagaagc gaggacaagc gggccaaagt
     1201 gacatctgcc atgcagacca tgctgttcac catgctgaga aagctggaca acgacgccct
     1261 gaacaacatc atcaacaacg ccagagatgg ctgcgtgccc ctgaatatca tccctctgac
     1321 aaccgccgcc aagctgatgg tggtcatccc cgactacaac acctacaaga atacctgcga
     1381 cggcaccacc ttcacctacg cctctgctct gtgggagatc cagcaggtcg tggacgccga
     1441 tagcaagatc gtgcagctga gcgagatcag catggacaac agccccaatc tggcctggcc
     1501 tctgattgtg acagccctga gagccaacag cgccgtgaaa ctgcaggcct aggagatatc
     1561 cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     1621 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     1681 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     1741 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     1801 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     1861 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     1921 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1981 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2041 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2101 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     2161 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     2221 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     2281 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     2341 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     2401 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     2461 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     2521 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     2581 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     2641 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     2701 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     2761 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     2821 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     2881 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     2941 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     3001 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     3061 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     3121 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     3181 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     3241 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3301 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3361 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3421 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3481 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3541 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3601 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3661 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3721 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3781 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3841 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3901 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3961 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4021 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4081 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4141 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4201 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4261 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4321 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4381 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4441 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4501 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4561 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4621 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4681 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4741 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4801 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4861 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4921 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4981 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5041 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5101 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5161 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5221 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5281 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5341 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5401 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5461 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5521 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5581 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5641 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     5701 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     5761 gttggaggtc gc
//
