LOCUS       sequence-307823-        6096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp5 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6096)
  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 6096)
  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-Nsp5
FEATURES             Location/Qualifiers
     source          1..6096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        135..514
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..718
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     669..689
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    720..738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    741..759
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     769..793
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             890..913
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             926..949
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1948..1965)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1954..2178
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2311..2330)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2462..2509
                     /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             2517..3539
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2517..3539
                     /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    3669..3790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3706..3725)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3760..3779
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3839..3855)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3839..3855)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3852..3874)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3863..3879
                     /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(3887..3893,3894..3911,3912..3917))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3932..3953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4070..4087)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4241..4829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4321..4340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5000..5791,5792..5860))
                     /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     5623..5642
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5861..5965)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6040..6059)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
       61 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      121 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      181 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      241 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      301 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      361 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      421 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      481 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      541 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      601 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      661 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      721 ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt
      781 gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg
      841 ggaccgatcc agcctccgga ctctagcgtt taaacttaag cttgagatgg actataaaga
      901 tgacgatgac aaggccgcag ccgcacatca ccaccatcat caccatcacg gatccgggat
      961 gagtgggttt cgaaagatgg ctttcccatc tggaaaggtc gagggttgca tggttcaagt
     1021 gacatgcggc actaccacat tgaacggact ctggttggat gatgttgtat attgtccccg
     1081 gcatgttata tgcacttctg aggacatgct gaatcctaat tacgaggact tgctgatacg
     1141 gaagagtaat cacaattttt tggttcaagc cggtaatgtt caactgcgcg tcattggcca
     1201 cagtatgcag aactgtgttt tgaagttgaa ggtggacacg gctaatccca aaactccgaa
     1261 atacaaattt gtgagaatcc aaccgggaca gacgttcagt gtcttggcat gttacaacgg
     1321 ttcccctagc ggagtatacc aatgcgctat gcgcccaaat ttcacaatca aaggcagttt
     1381 tctgaatggt tcctgtggct ctgtcggttt taacattgac tacgactgtg tttccttctg
     1441 ttacatgcat cacatggagc tcccaacggg agtacatgca ggtactgatc tcgaagggaa
     1501 tttttacggc ccatttgtgg accggcagac tgcacaggcg gcgggcactg acacgactat
     1561 tactgtaaac gttcttgcat ggctctacgc ggcagtaatt aacggtgata ggtggttcct
     1621 caatagattt acaactactt tgaatgattt caacctcgtc gctatgaagt ataattatga
     1681 gcccctgacg caggaccatg tagatatact gggacccttg agcgcacaaa caggcatagc
     1741 tgtactcgat atgtgcgcct ctctcaagga actgttgcag aatggcatga acggcaggac
     1801 gatactgggg tctgcgctgt tggaggatga gtttacgccg tttgacgttg ttcgccaatg
     1861 ttccggggtc accttccaag cctaggagat atccagcaca gtggcggccg ctcgagtcta
     1921 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1981 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2041 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2101 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2161 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2221 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2281 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2341 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2401 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     2461 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     2521 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     2581 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     2641 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     2701 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     2761 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     2821 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     2881 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     2941 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     3001 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     3061 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     3121 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     3181 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     3241 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     3301 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     3361 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     3421 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     3481 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     3541 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3601 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3661 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3721 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3781 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3841 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3901 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3961 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4021 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4081 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4141 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4201 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4261 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4321 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4381 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4441 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4501 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4561 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4621 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4681 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4741 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4801 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4861 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4921 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4981 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5041 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5101 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5161 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5221 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5281 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5341 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5401 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5461 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5521 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5581 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5641 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5701 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5761 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5821 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5881 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5941 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6001 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     6061 atagttaagc cagtatctgc tccctgcttg tgtgtt
//
