LOCUS       sequence-307614-        7038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged 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 7038)
  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 7038)
  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
FEATURES             Location/Qualifiers
     source          1..7038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        117..496
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        497..700
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     651..671
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    702..720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    723..741
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     751..775
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2872..2889)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2878..3102
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3235..3254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3386..3433
                     /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             3441..4463
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3441..4463
                     /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    4593..4714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4630..4649)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4684..4703
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4763..4779)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4763..4779)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4776..4798)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4787..4803
                     /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(4811..4817,4818..4835,4836..4841))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4856..4877
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4994..5011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5165..5753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5245..5264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5924..6715,6716..6784))
                     /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     6547..6566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6785..6889)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6964..6983)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
       61 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      121 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      181 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      241 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      301 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      361 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      421 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      481 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      541 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      601 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      661 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga
      721 tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg
      781 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
      841 gactctagcg tttaaactta agcttggtac cgagctcgga tccgggatgg catacacacg
      901 ctatgttgat aataactttt gtggcccaga cggctatccg ctcgaatgca taaaggacct
      961 tcttgcacgg gccggaaaag cttcctgcac tttgtctgag cagttggact tcatcgatac
     1021 caaaagagga gtttattgct gccgagagca cgagcacgaa atcgcgtggt acactgagcg
     1081 ctccgagaaa agctacgaat tgcagacgcc tttcgaaatt aaactcgcaa agaaattcga
     1141 caccttcaat ggtgagtgcc caaattttgt cttcccactt aacagtatta tcaagaccat
     1201 acagccgaga gtggagaaaa agaagctgga cggtttcatg ggtcggataa ggagcgtata
     1261 ccctgttgcg tcccccaacg aatgcaacca aatgtgtttg tcaacgctca tgaaatgcga
     1321 ccattgtggt gagactagct ggcaaactgg tgacttcgtc aaagctactt gcgaattttg
     1381 tggcacggaa aaccttacca aagaaggcgc tactacttgt gggtaccttc cgcagaatgc
     1441 ggtggtaaaa atctattgcc ctgcgtgcca caactctgaa gtcggacccg aacattctct
     1501 cgctgaatat cacaacgaat ctggtctcaa aacgattctt cgaaaaggcg gacgcacaat
     1561 tgcgtttggc ggctgcgtgt tttcttacgt cggctgtcat aacaaatgcg cctactgggt
     1621 gccgcgggct tcagccaata ttggttgtaa ccataccgga gtggtcgggg aagggagcga
     1681 gggtctgaat gataacctgt tggaaatttt gcaaaaggaa aaggtaaaca ttaatatcgt
     1741 tggagatttt aaattgaacg aagaaatcgc gattattctg gcatcctttt cagcatccac
     1801 cagtgccttc gtagagactg tgaagggact tgactacaag gcttttaagc agatagtcga
     1861 gtcttgtggg aatttcaagg taacaaaagg taaagcgaag aagggtgctt ggaatattgg
     1921 tgaacagaaa agcatactca gcccattgta cgcatttgcc agtgaggcgg cacgagtagt
     1981 gcgaagtatt ttcagcagga cgctcgaaac agcgcagaac tccgtcaggg tgctccaaaa
     2041 agccgccatt actatccttg atggaattag tcaatacagc ttgagactga tagacgcgat
     2101 gatgtttacc tccgacttgg caacgaataa cttggtggtg atggcgtata ttacgggcgg
     2161 cgttgttcaa cttacatcac agtggctcac aaatatattt gggacggtct acgagaagct
     2221 taagccggtt ctcgactggc tggaagaaaa gtttaaagag ggggttgaat ttctccgaga
     2281 tggatgggaa attgtcaaat ttatttccac atgtgcctgc gagatagtag gcgggcaaat
     2341 tgtcacatgc gcgaaagaaa ttaaagaaag cgttcaaaca tttttcaagc tcgtgaataa
     2401 gttcttggct ctgtgtgctg acagcataat aataggcggg gctaagctga aagcgttgaa
     2461 tctcggggaa acatttgtta cgcatagtaa agggctttat cggaagtgtg tgaaaagtag
     2521 ggaagaaacc ggcctgctca tgccgttgaa ggctccaaaa gagataatat ttctggaggg
     2581 cgaaactctg cccaccgagg ttctgaccga ggaagtggta ctcaaaactg gggacctgca
     2641 gcctcttgag caacccacgt ccgaggctgt tgaggcccct cttgttggca cacccgtgtg
     2701 cataaacggt ttgatgctct tggagatcaa ggacacagaa aagtattgtg cactcgcgcc
     2761 caatatgatg gttacgaata atacctttac gctcaaggga ggtgcctagg agatatccag
     2821 cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2881 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2941 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3001 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3061 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3121 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3181 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3241 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3301 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3361 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     3421 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     3481 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     3541 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     3601 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     3661 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     3721 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     3781 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3841 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     3901 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3961 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     4021 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     4081 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     4141 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     4201 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     4261 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     4321 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     4381 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     4441 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     4501 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4561 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4621 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4681 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4741 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4801 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4861 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4921 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4981 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5041 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5101 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5161 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5221 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5281 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5341 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5401 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5461 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5521 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5581 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5641 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5701 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5761 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5821 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5881 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5941 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6001 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6061 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6121 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6181 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6241 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6301 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6361 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6421 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6481 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6541 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6601 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6661 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6721 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6781 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6841 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6901 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6961 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     7021 ggaggtcgct gagtagtg
//
