LOCUS       sequence-307621-        7082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 
            Nsp2 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7082)
  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 7082)
  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-Nsp2-HF
FEATURES             Location/Qualifiers
     source          1..7082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        187..566
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        567..770
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     721..741
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    772..790
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    793..811
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     821..845
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2886..2909
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2922..2945
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2986..3003)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2992..3216
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3349..3368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3500..3547
                     /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             3555..4577
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3555..4577
                     /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    4707..4828
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4744..4763)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4798..4817
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4877..4893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4877..4893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4890..4912)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4901..4917
                     /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(4925..4931,4932..4949,4950..4955))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4970..4991
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5108..5125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5279..5867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5359..5378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6038..6829,6830..6898))
                     /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     6661..6680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6899..7003)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(7078..7082,1..15))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
       61 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      121 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      181 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      241 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      301 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      361 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      421 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      481 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      541 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      601 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      661 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      721 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
      781 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
      841 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      901 ccagcctccg gactctagcg tttaaactta agcttggtac cgagctcgga tccgggatgg
      961 catacacacg ctatgttgat aataactttt gtggcccaga cggctatccg ctcgaatgca
     1021 taaaggacct tcttgcacgg gccggaaaag cttcctgcac tttgtctgag cagttggact
     1081 tcatcgatac caaaagagga gtttattgct gccgagagca cgagcacgaa atcgcgtggt
     1141 acactgagcg ctccgagaaa agctacgaat tgcagacgcc tttcgaaatt aaactcgcaa
     1201 agaaattcga caccttcaat ggtgagtgcc caaattttgt cttcccactt aacagtatta
     1261 tcaagaccat acagccgaga gtggagaaaa agaagctgga cggtttcatg ggtcggataa
     1321 ggagcgtata ccctgttgcg tcccccaacg aatgcaacca aatgtgtttg tcaacgctca
     1381 tgaaatgcga ccattgtggt gagactagct ggcaaactgg tgacttcgtc aaagctactt
     1441 gcgaattttg tggcacggaa aaccttacca aagaaggcgc tactacttgt gggtaccttc
     1501 cgcagaatgc ggtggtaaaa atctattgcc ctgcgtgcca caactctgaa gtcggacccg
     1561 aacattctct cgctgaatat cacaacgaat ctggtctcaa aacgattctt cgaaaaggcg
     1621 gacgcacaat tgcgtttggc ggctgcgtgt tttcttacgt cggctgtcat aacaaatgcg
     1681 cctactgggt gccgcgggct tcagccaata ttggttgtaa ccataccgga gtggtcgggg
     1741 aagggagcga gggtctgaat gataacctgt tggaaatttt gcaaaaggaa aaggtaaaca
     1801 ttaatatcgt tggagatttt aaattgaacg aagaaatcgc gattattctg gcatcctttt
     1861 cagcatccac cagtgccttc gtagagactg tgaagggact tgactacaag gcttttaagc
     1921 agatagtcga gtcttgtggg aatttcaagg taacaaaagg taaagcgaag aagggtgctt
     1981 ggaatattgg tgaacagaaa agcatactca gcccattgta cgcatttgcc agtgaggcgg
     2041 cacgagtagt gcgaagtatt ttcagcagga cgctcgaaac agcgcagaac tccgtcaggg
     2101 tgctccaaaa agccgccatt actatccttg atggaattag tcaatacagc ttgagactga
     2161 tagacgcgat gatgtttacc tccgacttgg caacgaataa cttggtggtg atggcgtata
     2221 ttacgggcgg cgttgttcaa cttacatcac agtggctcac aaatatattt gggacggtct
     2281 acgagaagct taagccggtt ctcgactggc tggaagaaaa gtttaaagag ggggttgaat
     2341 ttctccgaga tggatgggaa attgtcaaat ttatttccac atgtgcctgc gagatagtag
     2401 gcgggcaaat tgtcacatgc gcgaaagaaa ttaaagaaag cgttcaaaca tttttcaagc
     2461 tcgtgaataa gttcttggct ctgtgtgctg acagcataat aataggcggg gctaagctga
     2521 aagcgttgaa tctcggggaa acatttgtta cgcatagtaa agggctttat cggaagtgtg
     2581 tgaaaagtag ggaagaaacc ggcctgctca tgccgttgaa ggctccaaaa gagataatat
     2641 ttctggaggg cgaaactctg cccaccgagg ttctgaccga ggaagtggta ctcaaaactg
     2701 gggacctgca gcctcttgag caacccacgt ccgaggctgt tgaggcccct cttgttggca
     2761 cacccgtgtg cataaacggt ttgatgctct tggagatcaa ggacacagaa aagtattgtg
     2821 cactcgcgcc caatatgatg gttacgaata atacctttac gctcaaggga ggtgcatcgg
     2881 gcggacatca ccaccatcat caccatcacg ccgcagccgc agattataaa gatgacgatg
     2941 acaagtagct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     3001 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3061 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3121 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3181 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3241 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3301 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3361 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3421 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3481 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     3541 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3601 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3661 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3721 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3781 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3841 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3901 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3961 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4021 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4081 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4141 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4201 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4261 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4321 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4381 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4441 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4501 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4561 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4621 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4681 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4741 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4801 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4861 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4921 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4981 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5041 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5101 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5161 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5221 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5281 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5341 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5401 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5461 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5521 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5581 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5641 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5701 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5761 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5821 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5881 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5941 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6001 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6061 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6121 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6181 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6241 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6301 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6361 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6421 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6481 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6541 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6601 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6661 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6721 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6781 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6841 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6901 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6961 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7021 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     7081 aa
//
