LOCUS       sequence-307800-        6042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged 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 6042)
  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 6042)
  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-Nsp5
FEATURES             Location/Qualifiers
     source          1..6042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        131..510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     665..685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    716..734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    737..755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     765..789
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(1890..1907)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1896..2120
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2253..2272)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2404..2451
                     /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             2459..3481
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2459..3481
                     /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    3611..3732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3648..3667)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3702..3721
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3781..3797)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3781..3797)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3794..3816)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3805..3821
                     /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(3829..3835,3836..3853,3854..3859))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3874..3895
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4012..4029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4183..4771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4263..4282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4942..5733,5734..5802))
                     /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     5565..5584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5803..5907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5982..6001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
       61 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      121 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      481 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct
      721 atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac
      781 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
      841 cgatccagcc tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccggg
      901 atgagtgggt ttcgaaagat ggctttccca tctggaaagg tcgagggttg catggttcaa
      961 gtgacatgcg gcactaccac attgaacgga ctctggttgg atgatgttgt atattgtccc
     1021 cggcatgtta tatgcacttc tgaggacatg ctgaatccta attacgagga cttgctgata
     1081 cggaagagta atcacaattt tttggttcaa gccggtaatg ttcaactgcg cgtcattggc
     1141 cacagtatgc agaactgtgt tttgaagttg aaggtggaca cggctaatcc caaaactccg
     1201 aaatacaaat ttgtgagaat ccaaccggga cagacgttca gtgtcttggc atgttacaac
     1261 ggttccccta gcggagtata ccaatgcgct atgcgcccaa atttcacaat caaaggcagt
     1321 tttctgaatg gttcctgtgg ctctgtcggt tttaacattg actacgactg tgtttccttc
     1381 tgttacatgc atcacatgga gctcccaacg ggagtacatg caggtactga tctcgaaggg
     1441 aatttttacg gcccatttgt ggaccggcag actgcacagg cggcgggcac tgacacgact
     1501 attactgtaa acgttcttgc atggctctac gcggcagtaa ttaacggtga taggtggttc
     1561 ctcaatagat ttacaactac tttgaatgat ttcaacctcg tcgctatgaa gtataattat
     1621 gagcccctga cgcaggacca tgtagatata ctgggaccct tgagcgcaca aacaggcata
     1681 gctgtactcg atatgtgcgc ctctctcaag gaactgttgc agaatggcat gaacggcagg
     1741 acgatactgg ggtctgcgct gttggaggat gagtttacgc cgtttgacgt tgttcgccaa
     1801 tgttccgggg tcaccttcca agcctaggag atatccagca cagtggcggc cgctcgagtc
     1861 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1921 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1981 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2041 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2101 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2161 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2221 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2281 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2341 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     2401 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     2461 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     2521 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     2581 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     2641 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     2701 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     2761 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     2821 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     2881 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     2941 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3001 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3061 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     3121 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     3181 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     3241 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     3301 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     3361 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     3421 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     3481 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3541 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3601 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3661 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3721 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3781 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3841 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3901 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3961 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4021 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4081 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4141 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4201 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4261 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4321 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4381 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4441 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4501 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4561 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4621 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4681 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4741 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4801 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4861 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4921 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4981 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5041 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5101 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5161 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5221 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5281 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5341 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5401 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5461 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5521 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5581 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5641 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5701 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5761 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5821 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5881 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     5941 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6001 gcatagttaa gccagtatct gctccctgct tgtgtgttgg ag
//
