LOCUS       sequence-307940-        6375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged SARS-CoV-2 N protein under control 
            of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6375)
  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 6375)
  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-Nprot_GC3opt
FEATURES             Location/Qualifiers
     source          1..6375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        98..477
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        478..681
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     632..652
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    683..701
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    704..722
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     732..756
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2190..2207)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2196..2420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2553..2572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2704..2751
                     /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             2759..3781
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2759..3781
                     /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    3911..4032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3948..3967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4002..4021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4081..4097)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4081..4097)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4094..4116)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4105..4121
                     /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(4129..4135,4136..4153,4154..4159))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4174..4195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4312..4329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4483..5071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4563..4582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5242..6033,6034..6102))
                     /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     5865..5884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6103..6207)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6282..6301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
       61 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
      121 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      181 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      241 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      301 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      361 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      421 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      481 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      541 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      601 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      661 gaggtctata taagcagagc tctccctatc agtgatagag atctccctat cagtgataga
      721 gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt
      781 tttgacctcc atagaagaca ccgggaccga tccagcctcc ggactctagc gtttaaactt
      841 aagcttggta ccgagctcgg atccgggatg agcgacaacg gccctcagaa ccagagaaac
      901 gcccctcgga tcacatttgg cggccctagc gatagcaccg gcagcaatca gaatggcgag
      961 agaagcggcg ccagaagcaa gcagagaagg cctcaaggcc tgcctaacaa caccgccagc
     1021 tggttcacag ccctgacaca gcacggcaaa gaggacctga agttccctag aggacagggc
     1081 gtgcccatca acaccaacag cagccccgat gaccagatcg gctactacag acgggccacc
     1141 agaagaatca gaggcggcga cggcaagatg aaggatctga gccccagatg gtacttctac
     1201 tacctcggca caggacccga agccggactt ccttatggcg ccaacaagga cggcatcatc
     1261 tgggttgcaa cagaaggcgc cctgaacacc cctaaggacc acatcggcac cagaaatccc
     1321 gccaacaatg ccgccattgt gctgcagttg cctcagggca caacactgcc caagggcttt
     1381 tacgccgagg gctctagagg cggatctcag gccagcagca gaagcagctc cagatccaga
     1441 aacagctccc ggaatagcac ccctggctcc agcagaggaa caagccctgc tagaatggcc
     1501 ggcaacggcg gagatgctgc tctggcactt ctcctgctgg accggctgaa tcagctggaa
     1561 agcaagatga gcggcaaggg acagcagcag cagggccaga ccgtgacaaa aaagtctgcc
     1621 gccgaggcca gcaagaagcc cagacagaaa agaaccgcca ccaaggccta caacgtgacc
     1681 caggcctttg gcagaagagg ccctgagcag acccagggca atttcggcga tcaagagctg
     1741 atcagacagg gcaccgacta caagcactgg cctcagatcg cccagtttgc cccatctgcc
     1801 agcgcctttt tcggcatgag ccggatcggc atggaagtga cacctagcgg cacctggctg
     1861 acatacacag gcgccatcaa gctggacgac aaggacccca acttcaagga ccaagtgatc
     1921 ctgctgaaca agcacatcga cgcctacaag acattccctc caaccgagcc taagaaggac
     1981 aagaagaaga aggccgacga gacacaggcc ctgcctcagc gccagaaaaa gcagcagaca
     2041 gtgacactgc tgccagccgc cgacctggac gatttttcta agcagctgca gcagagcatg
     2101 agcagcgccg attctacaca ggcctaggag atatccagca cagtggcggc cgctcgagtc
     2161 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2221 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2281 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2341 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2401 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2461 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2521 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2581 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2641 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     2701 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     2761 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     2821 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     2881 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     2941 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     3001 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     3061 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     3121 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     3181 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     3241 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3301 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3361 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     3421 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     3481 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     3541 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     3601 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     3661 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     3721 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     3781 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3841 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3901 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3961 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4021 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4081 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4141 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4201 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4261 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4321 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4381 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4441 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4501 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4561 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4621 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4681 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4741 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4801 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4861 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4921 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4981 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5041 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5101 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5161 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5221 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5281 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5341 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5401 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5461 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5521 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5581 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5641 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5701 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5761 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5821 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5881 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5941 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6001 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6061 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6121 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6181 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6241 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6301 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     6361 agcaaaattt aagct
//
