LOCUS       sequence-307916-        6162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged SARS-CoV-2 Nsp15 under control of a
            tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6162)
  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 6162)
  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-Nsp15
FEATURES             Location/Qualifiers
     source          1..6162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        121..500
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        501..704
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     655..675
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    706..724
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    727..745
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     755..779
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2000..2017)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2006..2230
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2363..2382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2514..2561
                     /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             2569..3591
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2569..3591
                     /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    3721..3842
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3758..3777)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3812..3831
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3891..3907)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3891..3907)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3904..3926)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3915..3931
                     /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(3939..3945,3946..3963,3964..3969))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3984..4005
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4122..4139)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4293..4881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4373..4392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5052..5843,5844..5912))
                     /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     5675..5694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5913..6017)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6092..6111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
       61 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      121 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      181 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      241 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      301 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      361 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      421 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      481 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      541 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      601 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      661 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      721 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      781 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      841 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccggg atgagtctgg
      901 aaaacgtcgc ttttaatgtc gtaaataagg ggcattttga tggtcaacag ggggaagttc
      961 ctgtttccat aatcaacaac actgtgtata ctaaagtaga tggtgtggat gtcgaacttt
     1021 ttgaaaataa gacgaccctg ccggtaaacg tggcgttcga attgtgggcg aagaggaata
     1081 taaaaccagt accggaagta aagatcttga acaatcttgg tgtggatata gcggccaata
     1141 ccgtgatatg ggattataaa agggatgcac ctgctcacat cagtactatt ggtgtctgct
     1201 ccatgacgga catcgctaaa aagcctaccg agacgatctg cgctccgctg actgtgttct
     1261 ttgatggaag ggtcgatggg caagtggacc tgtttcggaa cgcaaggaac ggggttctca
     1321 taactgaagg gtccgtcaag gggcttcagc catcagtagg acctaaacag gcttcactca
     1381 atggcgtgac gcttattggc gaggcggtga agactcagtt taactactat aaaaaagtag
     1441 atggcgtagt acaacaactg cccgaaacgt attttacgca gtcacgcaac ctgcaagagt
     1501 tcaagccacg ctcacagatg gagatcgact tccttgagct tgcgatggac gagttcattg
     1561 agagatacaa gctcgaaggg tatgcgtttg aacatatagt atatggagac ttctcccata
     1621 gtcagctggg tgggcttcac ctgctcatcg ggctggcgaa gcgattcaaa gaatctccct
     1681 tcgaactcga agactttatc ccaatggact ccacggtgaa gaactacttt attacagacg
     1741 cccaaactgg tagttctaaa tgcgtgtgct cagttataga cctgctgctt gatgactttg
     1801 tagaaataat aaaatcacag gacctttcag tcgtgtccaa ggtggttaag gtcactatag
     1861 actatacaga aatatctttc atgctgtggt gcaaggacgg acacgtggaa actttctacc
     1921 ctaagttgca agcctaggag atatccagca cagtggcggc cgctcgagtc tagagggccc
     1981 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2041 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2101 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2161 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2221 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2281 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2341 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2401 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2461 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     2521 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     2581 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     2641 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     2701 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     2761 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     2821 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     2881 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     2941 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     3001 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     3061 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     3121 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     3181 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     3241 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     3301 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     3361 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     3421 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     3481 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     3541 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     3601 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3661 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3721 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3781 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3841 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     3901 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3961 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4021 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4081 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4141 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4201 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4261 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4321 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4381 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4441 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4501 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4561 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4621 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4681 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4741 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4801 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4861 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4921 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4981 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5041 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5101 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5161 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5221 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5281 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5341 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5401 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5461 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5521 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5581 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5641 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5701 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5761 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5821 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5881 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5941 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6001 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     6061 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     6121 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gt
//
