LOCUS       sequence-307937-        6381 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 6381)
  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 6381)
  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-Nprot
FEATURES             Location/Qualifiers
     source          1..6381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        112..491
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        492..695
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     646..666
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    697..715
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    718..736
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     746..770
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2210..2227)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2216..2440
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2573..2592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2724..2771
                     /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             2779..3801
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2779..3801
                     /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    3931..4052
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3968..3987)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4022..4041
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4101..4117)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4101..4117)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4114..4136)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4125..4141
                     /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(4149..4155,4156..4173,4174..4179))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4194..4215
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4332..4349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4503..5091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4583..4602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5262..6053,6054..6122))
                     /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     5885..5904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6123..6227)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6302..6321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
       61 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      121 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      181 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      241 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      301 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      361 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      421 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      481 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      541 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      601 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      661 ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat agagatctcc
      721 ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc gcctggagac
      781 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc
      841 tagcgtttaa acttaagctt ggtaccgagc tcggatccgg gatgtccgac aatggtccgc
      901 agaaccagag aaacgcaccc aggataacat ttgggggtcc aagtgacagt acaggatcta
      961 accagaatgg ggagcgaagc ggggcacggt ctaagcaacg cagaccccag ggtcttccta
     1021 ataatacagc atcatggttt accgctctca cacaacacgg aaaagaagac ttgaagttcc
     1081 ctcgcgggca gggggtcccg atcaatacga atagttctcc cgatgatcaa atcggttatt
     1141 accgaagggc cacccgcaga attcgaggag gggatggtaa gatgaaggat cttagtccaa
     1201 ggtggtattt ctattacctc ggcacgggcc cggaggcggg cctcccatac ggggctaaca
     1261 aggacggaat catctgggtg gcgaccgagg gtgccctcaa cactcccaag gatcatatag
     1321 gaacgcggaa tccagcaaat aatgcggcta tagtgttgca acttccgcaa ggaacaaccc
     1381 tccctaaggg attctatgca gaggggtcca ggggaggttc tcaagctagt tctcgctcat
     1441 caagccgatc tcggaattca tctcggaatt caacgccagg atctagtcga ggcacaagtc
     1501 cggcccggat ggccggaaac ggcggcgatg ccgcgttggc actcctgctc ttggatcgcc
     1561 ttaaccaact tgaatctaaa atgagtggta aggggcagca acaacagggg cagacagtga
     1621 caaaaaagag tgcagcagag gcgagtaaga agcccaggca aaaacgcaca gcaacgaaag
     1681 catataacgt cacccaggca tttggtagac ggggccccga acagacacaa ggtaacttcg
     1741 gagatcagga actcatccgg caaggtaccg actacaagca ttggccacag atcgcgcagt
     1801 ttgctcccag tgcgtccgct ttcttcggaa tgtctcggat aggcatggag gttacgccga
     1861 gtggcacgtg gcttacatat accggagcaa tcaaactgga tgacaaagat ccaaatttca
     1921 aagatcaggt tatcttgttg aacaaacata ttgatgccta taaaacgttt ccacccacag
     1981 aacctaaaaa agacaagaag aaaaaagccg acgaaacgca agcccttcca cagaggcaga
     2041 aaaagcagca aaccgtcact ctgctgcctg ctgccgacct ggatgacttc tctaagcagc
     2101 tccaacaatc aatgtcaagt gccgattcaa cccaggccta agcctaggag atatccagca
     2161 cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     2221 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2281 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2341 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2401 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2461 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2521 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2581 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2641 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2701 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
     2761 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     2821 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     2881 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     2941 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     3001 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     3061 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     3121 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     3181 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     3241 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     3301 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     3361 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     3421 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     3481 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     3541 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     3601 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     3661 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     3721 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     3781 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     3841 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3901 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     3961 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4021 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4081 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4141 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4201 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4261 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4321 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4381 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4441 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4501 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4561 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4621 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4681 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4741 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4801 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4861 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4921 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4981 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5041 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5101 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5161 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5221 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5281 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5341 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5401 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5461 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5521 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5581 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5641 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5701 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5761 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5821 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5881 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5941 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6001 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6061 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6121 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6181 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6241 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     6301 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     6361 aggtcgctga gtagtgcgcg a
//
