LOCUS       sequence-307874-        7920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged SARS-CoV-2 Nsp12 under control of a
            tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7920)
  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 7920)
  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-Nsp12
FEATURES             Location/Qualifiers
     source          1..7920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..39)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        211..590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        591..794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     745..765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    796..814
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    817..835
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     845..869
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(3848..3865)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3854..4078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4211..4230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4362..4409
                     /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             4417..5439
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4417..5439
                     /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    5569..5690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5606..5625)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5660..5679
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5739..5755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5739..5755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5752..5774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5763..5779
                     /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(5787..5793,5794..5811,5812..5817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5832..5853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5970..5987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6141..6729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6221..6240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6900..7691,7692..7760))
                     /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     7523..7542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7761..7865)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
       61 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
      121 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
      181 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      241 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      301 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      361 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      421 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      481 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      541 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      601 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      661 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      721 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      781 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      841 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      901 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttg
      961 gtaccgagct cggatccggg atgtcagcgg atgcgcagtc ttttcttaat cgggtttgcg
     1021 gagtatctgc ggcccggctt accccgtgcg gaactggcac cagtaccgat gtggtataca
     1081 gggccttcga catatataac gataaggtcg cgggttttgc aaagtttctc aagactaatt
     1141 gctgtcgctt tcaggaaaaa gacgaagatg acaatttgat cgattcctat ttcgtggtta
     1201 agagacatac tttttctaac taccaacatg aagaaacaat atacaatctc ctcaaagatt
     1261 gtcccgccgt cgccaaacac gactttttta agtttcgaat cgatggcgat atggtcccgc
     1321 atatttctcg acagcggctc actaagtaca ctatggcgga ccttgtgtat gctctccgcc
     1381 actttgacga ggggaattgt gacacgctta aagagatcct tgtaacctat aactgctgtg
     1441 atgatgacta tttcaataag aaggactggt acgattttgt tgagaacccc gacatcctgc
     1501 gcgtgtatgc caatctcgga gagcgggtac ggcaagcgtt gctgaaaacc gtacaattct
     1561 gtgacgcgat gcgcaacgca ggaatcgtag gtgtgctcac actggacaat caggatctta
     1621 acggtaattg gtacgatttt ggagatttta tacagactac ccctggctcc ggtgtcccag
     1681 tagtggattc atattactcc ttgctcatgc ccatacttac ccttacgagg gctttgacgg
     1741 cagagagtca tgtagatact gaccttacga agccgtacat taaatgggac ctgttgaagt
     1801 atgattttac ggaagagagg ttgaagttgt tcgataggta ctttaaatac tgggatcaga
     1861 cttatcatcc aaattgcgtt aattgcttgg atgaccgatg cattttgcac tgtgcgaatt
     1921 tcaacgtgct cttttccacg gttttccccc cgacgtcctt tggaccactg gtcaggaaaa
     1981 tttttgtcga cggtgtaccc tttgtggtct ccacaggata ccattttcga gagcttggag
     2041 tcgtacacaa tcaagatgta aacctgcatt caagtcgcct ctcctttaaa gaactccttg
     2101 tctatgcagc cgatcctgca atgcacgcag cgtcagggaa cctcctcttg gacaaaagga
     2161 ccacgtgttt ctcagtcgca gcgttgacga ataacgtggc gttccagacc gtaaagcctg
     2221 gtaatttcaa caaagacttc tatgatttcg cggtcagtaa agggttcttc aaggaaggat
     2281 catcagttga gcttaagcac tttttttttg cccaagacgg caacgcagca atttccgatt
     2341 atgactacta tcgatacaac ctgcctacta tgtgtgacat tcgccagctt cttttcgtcg
     2401 ttgaagttgt agataagtac tttgattgct atgatggcgg ttgtattaat gcgaaccagg
     2461 tgatcgtcaa caatctcgat aaatctgccg ggtttccctt taacaaatgg ggcaaagcga
     2521 ggttgtatta cgattctatg tcctacgagg atcaggatgc cctcttcgca tacaccaagc
     2581 ggaatgtgat tcctaccatt acacaaatga acttgaaata cgccatatct gccaaaaacc
     2641 gcgctcgcac tgttgcaggt gtgtctatat gctccacgat gactaatagg cagtttcatc
     2701 agaagttgct caaaagtatc gcagccactc gaggggcaac ggttgtgata gggacgagta
     2761 agttttatgg cggctggcat aatatgctga agaccgtgta tagcgatgtt gagaatccac
     2821 acctcatggg ttgggattat ccgaaatgcg atcgcgctat gcctaatatg ctcagaatca
     2881 tggcaagtct tgtgttggct cggaaacaca caacttgctg ctctctttct cataggttct
     2941 atagacttgc taacgaatgt gcccaggttc tctccgagat ggttatgtgt ggcggtagct
     3001 tgtacgtaaa gccgggaggg acatcttctg gtgacgccac gactgcgtat gctaattctg
     3061 tattcaacat ctgtcaggca gtaactgcga acgttaacgc cttgctcagc acagacggca
     3121 acaagatagc ggataaatac gtgcgcaatc tgcagcacag gctgtatgag tgtttgtatc
     3181 gcaatcgaga tgtggacacc gacttcgtga atgagtttta tgcttatctc agaaaacact
     3241 tttctatgat gatactcagc gatgatgccg tggtctgttt taacagcact tacgcaagcc
     3301 aaggccttgt tgcgagtatc aaaaatttta agagcgtgtt gtattaccaa aataatgtat
     3361 tcatgagcga ggccaaatgc tggacagaga ccgacctcac aaaggggcca cacgagtttt
     3421 gctcccagca cacgatgctg gtcaaacagg gagatgacta tgtatacctg ccgtatcccg
     3481 atccgtctcg gattctgggg gcgggctgct ttgtcgatga catagtgaag acggacggca
     3541 cacttatgat cgaacggttt gtttcactgg caatagatgc gtaccctttg actaaacatc
     3601 ccaaccagga atatgcggat gtgttccatc tttacttgca atatattagg aagttgcacg
     3661 acgagctgac ggggcatatg cttgatatgt actcagtcat gcttacgaat gataatacct
     3721 cacgctattg ggagccagaa ttctatgagg cgatgtatac accacacaca gtgctgcagg
     3781 cctaggagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc
     3841 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3901 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3961 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4021 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     4081 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     4141 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4201 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4261 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4321 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     4381 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     4441 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     4501 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     4561 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     4621 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     4681 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     4741 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     4801 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     4861 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     4921 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     4981 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     5041 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     5101 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     5161 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     5221 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     5281 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     5341 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     5401 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     5461 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5521 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     5581 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5641 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     5701 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5761 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5821 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5881 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5941 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6001 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6061 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6121 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6181 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6241 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6301 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6361 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6421 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6481 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6541 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6601 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6661 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6721 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6781 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6841 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6901 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6961 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7021 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7081 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7141 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7201 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7261 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7321 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7381 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7441 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7501 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7561 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7621 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7681 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7741 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7801 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7861 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
//
