LOCUS       sequence-307865-        6057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp8-Nsp7 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-Nsp8-Nsp7_GC3opt
FEATURES             Location/Qualifiers
     source          1..6057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        102..481
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        482..685
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     636..656
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    687..705
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    708..726
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     736..760
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             857..880
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             893..916
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1876..1893)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1882..2106
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2239..2258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2390..2437
                     /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             2445..3467
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2445..3467
                     /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    3597..3718
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3634..3653)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3688..3707
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3767..3783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3767..3783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3780..3802)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3791..3807
                     /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(3815..3821,3822..3839,3840..3845))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3860..3881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3998..4015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4169..4757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4249..4268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4928..5719,5720..5788))
                     /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     5551..5570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5789..5893)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5968..5987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
       61 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      121 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      181 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      241 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      301 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      361 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      421 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      481 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      541 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      601 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      661 gtgggaggtc tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga
      721 tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg
      781 ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa
      841 acttaagctt gagatggact ataaagatga cgatgacaag gccgcagccg cacatcacca
      901 ccatcatcac catcacggat ccgggatggc cattgccagc gagtttagca gcctgcctag
      961 ctacgccgcc tttgccacag ctcaagaggc ctatgaacag gccgtggcca atggcgattc
     1021 tgaggtggtg ctgaagaagc tgaaaaagtc cctgaacgtg gccaagagcg agttcgacag
     1081 agatgccgcc atgcagcgga agctggaaaa gatggccgac caggccatga cacagatgta
     1141 caagcaggcc agaagcgagg acaagcgggc caaagtgaca tctgccatgc agaccatgct
     1201 gttcaccatg ctgagaaagc tggacaacga cgccctgaac aacatcatca acaacgccag
     1261 agatggctgc gtgcccctga atatcatccc tctgacaacc gccgccaagc tgatggtggt
     1321 catccccgac tacaacacct acaagaatac ctgcgacggc accaccttca cctacgcctc
     1381 tgctctgtgg gagatccagc aggtcgtgga cgccgatagc aagatcgtgc agctgagcga
     1441 gatcagcatg gacaacagcc ccaatctggc ctggcctctg attgtgacag ccctgagagc
     1501 caacagcgcc gtgaaactgc aaggctctgc aggatctgct gccggctctg gcgagttcag
     1561 caagatgagc gacgtgaagt gcaccagcgt ggtgctgctg tctgttctgc agcagctgag
     1621 agtggaaagc agcagcaaac tgtgggccca gtgtgtccag ctgcacaatg acatcctgct
     1681 ggccaaggat accaccgagg ccttcgagaa gatggtgtcc ctgctgagtg tgctgctgag
     1741 catgcaaggc gccgtggaca tcaacaagct gtgcgaggaa atgctggata accgggccac
     1801 actgcaggcc taggagatat ccagcacagt ggcggccgct cgagtctaga gggcccgttt
     1861 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     1921 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     1981 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2041 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2101 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2161 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2221 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2281 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2341 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     2401 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     2461 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     2521 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     2581 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     2641 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     2701 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     2761 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     2821 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
     2881 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     2941 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     3001 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     3061 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     3121 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     3181 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     3241 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     3301 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     3361 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     3421 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
     3481 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3541 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     3601 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3661 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3721 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3781 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3841 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3901 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3961 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4021 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4081 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4141 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4201 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4261 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4321 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4381 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4441 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4501 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4561 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4621 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4681 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4741 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4801 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4861 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4921 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4981 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5041 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5101 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5161 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5221 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5281 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5341 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5401 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5461 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5521 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5581 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5641 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5701 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5761 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5821 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5881 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     5941 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6001 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaattta
//
