LOCUS       sequence-307827-        5427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp7 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5427)
  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 5427)
  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-Nsp7
FEATURES             Location/Qualifiers
     source          1..5427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        443..646
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     597..617
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    648..666
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    669..687
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     697..721
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             818..841
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             854..877
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1207..1224)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1213..1437
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1570..1589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1721..1768
                     /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             1776..2798
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1776..2798
                     /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    2928..3049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2965..2984)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3019..3038
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3098..3114)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3098..3114)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3111..3133)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3122..3138
                     /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(3146..3152,3153..3170,3171..3176))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3191..3212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3329..3346)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3500..4088)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3580..3599)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4259..5050,5051..5119))
                     /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     4882..4901
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5120..5224)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5299..5318)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
       61 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      121 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      181 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      241 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      301 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      361 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      421 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      481 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      541 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      601 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
      661 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
      721 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      781 cctccggact ctagcgttta aacttaagct tgagatggac tataaagatg acgatgacaa
      841 ggccgcagcc gcacatcacc accatcatca ccatcacgga tccgggatgt ctaagatgag
      901 tgatgtgaaa tgtacgtccg tggtgttgct ttctgtcctg caacagctta gagttgaatc
      961 tagcagtaag ctctgggcac aatgcgtgca actccacaat gatattctgt tggcgaaaga
     1021 tactactgaa gccttcgaga agatggtatc cttgttgtct gtccttttga gcatgcaagg
     1081 cgcggttgac atcaacaagt tgtgtgagga gatgctggat aaccgagcta cactgcaagc
     1141 ctaggagata tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct
     1201 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1261 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1321 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1381 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     1441 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     1501 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1561 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1621 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1681 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     1741 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     1801 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     1861 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     1921 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     1981 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     2041 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     2101 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     2161 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     2221 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     2281 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     2341 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     2401 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     2461 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     2521 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     2581 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     2641 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     2701 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     2761 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     2821 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     2881 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     2941 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3001 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3061 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3121 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3181 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3241 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3301 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3361 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3421 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3481 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3541 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3601 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3661 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3721 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3781 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3841 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3901 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3961 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4021 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4081 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4141 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4201 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4261 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4321 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4381 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4441 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4501 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4561 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4621 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4681 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4741 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4801 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4861 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4921 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4981 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5041 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5101 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5161 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5221 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5281 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5341 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5401 gcaaggcttg accgacaatt gcatgaa
//
