LOCUS       sequence-307938-        6435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged 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 6435)
  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 6435)
  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-Nprot
FEATURES             Location/Qualifiers
     source          1..6435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        56..435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        436..639
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     590..610
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    641..659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    662..680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     690..714
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             811..834
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             847..870
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2208..2225)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2214..2438
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2571..2590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2722..2769
                     /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             2777..3799
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2777..3799
                     /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    3929..4050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3966..3985)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4020..4039
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4099..4115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4099..4115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4112..4134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4123..4139
                     /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(4147..4153,4154..4171,4172..4177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4192..4213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4501..5089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4581..4600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5260..6051,6052..6120))
                     /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     5883..5902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6121..6225)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6300..6319)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
       61 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      121 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      181 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      241 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      301 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      361 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      421 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      481 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      541 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      601 ggtaggcgtg tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat
      661 ctccctatca gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg
      721 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      781 actctagcgt ttaaacttaa gcttgagatg gactataaag atgacgatga caaggccgca
      841 gccgcacatc accaccatca tcaccatcac ggatccggga tgtccgacaa tggtccgcag
      901 aaccagagaa acgcacccag gataacattt gggggtccaa gtgacagtac aggatctaac
      961 cagaatgggg agcgaagcgg ggcacggtct aagcaacgca gaccccaggg tcttcctaat
     1021 aatacagcat catggtttac cgctctcaca caacacggaa aagaagactt gaagttccct
     1081 cgcgggcagg gggtcccgat caatacgaat agttctcccg atgatcaaat cggttattac
     1141 cgaagggcca cccgcagaat tcgaggaggg gatggtaaga tgaaggatct tagtccaagg
     1201 tggtatttct attacctcgg cacgggcccg gaggcgggcc tcccatacgg ggctaacaag
     1261 gacggaatca tctgggtggc gaccgagggt gccctcaaca ctcccaagga tcatatagga
     1321 acgcggaatc cagcaaataa tgcggctata gtgttgcaac ttccgcaagg aacaaccctc
     1381 cctaagggat tctatgcaga ggggtccagg ggaggttctc aagctagttc tcgctcatca
     1441 agccgatctc ggaattcatc tcggaattca acgccaggat ctagtcgagg cacaagtccg
     1501 gcccggatgg ccggaaacgg cggcgatgcc gcgttggcac tcctgctctt ggatcgcctt
     1561 aaccaacttg aatctaaaat gagtggtaag gggcagcaac aacaggggca gacagtgaca
     1621 aaaaagagtg cagcagaggc gagtaagaag cccaggcaaa aacgcacagc aacgaaagca
     1681 tataacgtca cccaggcatt tggtagacgg ggccccgaac agacacaagg taacttcgga
     1741 gatcaggaac tcatccggca aggtaccgac tacaagcatt ggccacagat cgcgcagttt
     1801 gctcccagtg cgtccgcttt cttcggaatg tctcggatag gcatggaggt tacgccgagt
     1861 ggcacgtggc ttacatatac cggagcaatc aaactggatg acaaagatcc aaatttcaaa
     1921 gatcaggtta tcttgttgaa caaacatatt gatgcctata aaacgtttcc acccacagaa
     1981 cctaaaaaag acaagaagaa aaaagccgac gaaacgcaag cccttccaca gaggcagaaa
     2041 aagcagcaaa ccgtcactct gctgcctgct gccgacctgg atgacttctc taagcagctc
     2101 caacaatcaa tgtcaagtgc cgattcaacc caggcctaag cctaggagat atccagcaca
     2161 gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     2221 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2281 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2341 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2401 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2461 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2521 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2581 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2641 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2701 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     2761 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     2821 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     2881 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     2941 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     3001 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     3061 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     3121 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     3181 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     3241 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     3301 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     3361 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     3421 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     3481 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     3541 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     3601 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     3661 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     3721 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     3781 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     3841 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     3901 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     3961 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4021 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4081 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4261 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4921 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6241 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     6301 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     6361 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     6421 tgcatgaaga atctg
//
