LOCUS       sequence-307876-        7974 bp    DNA     circular SYN 14-APR-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged 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 7974)
  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 7974)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Nsp12
FEATURES             Location/Qualifiers
     source          1..7974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        124..503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        504..707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     658..678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    709..727
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    730..748
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     758..782
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             879..902
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             915..938
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3815..3832)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3821..4045
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4178..4197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4329..4376
                     /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             4384..5406
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4384..5406
                     /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    5536..5657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5573..5592)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5627..5646
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5706..5722)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5706..5722)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5719..5741)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5730..5746
                     /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(5754..5760,5761..5778,5779..5784))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5799..5820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5937..5954)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6108..6696)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6188..6207)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6867..7658,7659..7727))
                     /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     7490..7509
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7728..7832)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7907..7926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
       61 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      121 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      181 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      241 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      301 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      361 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      421 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      481 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      541 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      601 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      661 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      721 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      781 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      841 gcctccggac tctagcgttt aaacttaagc ttgagatgga ctataaagat gacgatgaca
      901 aggccgcagc cgcacatcac caccatcatc accatcacgg atccgggatg tcagcggatg
      961 cgcagtcttt tcttaatcgg gtttgcggag tatctgcggc ccggcttacc ccgtgcggaa
     1021 ctggcaccag taccgatgtg gtatacaggg ccttcgacat atataacgat aaggtcgcgg
     1081 gttttgcaaa gtttctcaag actaattgct gtcgctttca ggaaaaagac gaagatgaca
     1141 atttgatcga ttcctatttc gtggttaaga gacatacttt ttctaactac caacatgaag
     1201 aaacaatata caatctcctc aaagattgtc ccgccgtcgc caaacacgac ttttttaagt
     1261 ttcgaatcga tggcgatatg gtcccgcata tttctcgaca gcggctcact aagtacacta
     1321 tggcggacct tgtgtatgct ctccgccact ttgacgaggg gaattgtgac acgcttaaag
     1381 agatccttgt aacctataac tgctgtgatg atgactattt caataagaag gactggtacg
     1441 attttgttga gaaccccgac atcctgcgcg tgtatgccaa tctcggagag cgggtacggc
     1501 aagcgttgct gaaaaccgta caattctgtg acgcgatgcg caacgcagga atcgtaggtg
     1561 tgctcacact ggacaatcag gatcttaacg gtaattggta cgattttgga gattttatac
     1621 agactacccc tggctccggt gtcccagtag tggattcata ttactccttg ctcatgccca
     1681 tacttaccct tacgagggct ttgacggcag agagtcatgt agatactgac cttacgaagc
     1741 cgtacattaa atgggacctg ttgaagtatg attttacgga agagaggttg aagttgttcg
     1801 ataggtactt taaatactgg gatcagactt atcatccaaa ttgcgttaat tgcttggatg
     1861 accgatgcat tttgcactgt gcgaatttca acgtgctctt ttccacggtt ttccccccga
     1921 cgtcctttgg accactggtc aggaaaattt ttgtcgacgg tgtacccttt gtggtctcca
     1981 caggatacca ttttcgagag cttggagtcg tacacaatca agatgtaaac ctgcattcaa
     2041 gtcgcctctc ctttaaagaa ctccttgtct atgcagccga tcctgcaatg cacgcagcgt
     2101 cagggaacct cctcttggac aaaaggacca cgtgtttctc agtcgcagcg ttgacgaata
     2161 acgtggcgtt ccagaccgta aagcctggta atttcaacaa agacttctat gatttcgcgg
     2221 tcagtaaagg gttcttcaag gaaggatcat cagttgagct taagcacttt ttttttgccc
     2281 aagacggcaa cgcagcaatt tccgattatg actactatcg atacaacctg cctactatgt
     2341 gtgacattcg ccagcttctt ttcgtcgttg aagttgtaga taagtacttt gattgctatg
     2401 atggcggttg tattaatgcg aaccaggtga tcgtcaacaa tctcgataaa tctgccgggt
     2461 ttccctttaa caaatggggc aaagcgaggt tgtattacga ttctatgtcc tacgaggatc
     2521 aggatgccct cttcgcatac accaagcgga atgtgattcc taccattaca caaatgaact
     2581 tgaaatacgc catatctgcc aaaaaccgcg ctcgcactgt tgcaggtgtg tctatatgct
     2641 ccacgatgac taataggcag tttcatcaga agttgctcaa aagtatcgca gccactcgag
     2701 gggcaacggt tgtgataggg acgagtaagt tttatggcgg ctggcataat atgctgaaga
     2761 ccgtgtatag cgatgttgag aatccacacc tcatgggttg ggattatccg aaatgcgatc
     2821 gcgctatgcc taatatgctc agaatcatgg caagtcttgt gttggctcgg aaacacacaa
     2881 cttgctgctc tctttctcat aggttctata gacttgctaa cgaatgtgcc caggttctct
     2941 ccgagatggt tatgtgtggc ggtagcttgt acgtaaagcc gggagggaca tcttctggtg
     3001 acgccacgac tgcgtatgct aattctgtat tcaacatctg tcaggcagta actgcgaacg
     3061 ttaacgcctt gctcagcaca gacggcaaca agatagcgga taaatacgtg cgcaatctgc
     3121 agcacaggct gtatgagtgt ttgtatcgca atcgagatgt ggacaccgac ttcgtgaatg
     3181 agttttatgc ttatctcaga aaacactttt ctatgatgat actcagcgat gatgccgtgg
     3241 tctgttttaa cagcacttac gcaagccaag gccttgttgc gagtatcaaa aattttaaga
     3301 gcgtgttgta ttaccaaaat aatgtattca tgagcgaggc caaatgctgg acagagaccg
     3361 acctcacaaa ggggccacac gagttttgct cccagcacac gatgctggtc aaacagggag
     3421 atgactatgt atacctgccg tatcccgatc cgtctcggat tctgggggcg ggctgctttg
     3481 tcgatgacat agtgaagacg gacggcacac ttatgatcga acggtttgtt tcactggcaa
     3541 tagatgcgta ccctttgact aaacatccca accaggaata tgcggatgtg ttccatcttt
     3601 acttgcaata tattaggaag ttgcacgacg agctgacggg gcatatgctt gatatgtact
     3661 cagtcatgct tacgaatgat aatacctcac gctattggga gccagaattc tatgaggcga
     3721 tgtatacacc acacacagtg ctgcaggcct aggagatatc cagcacagtg gcggccgctc
     3781 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     3841 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3901 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3961 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4021 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     4081 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4141 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4201 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4261 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4321 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     4381 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     4441 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     4501 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     4561 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     4621 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     4681 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     4741 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     4801 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     4861 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     4921 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     4981 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     5041 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     5101 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     5161 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     5221 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     5281 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     5341 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     5401 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5461 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5521 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5581 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5641 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     5701 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5761 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5821 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5881 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5941 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6001 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6061 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6121 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6181 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6241 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6301 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6361 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6421 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6481 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6541 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6601 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6661 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6721 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6781 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6841 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6901 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6961 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7021 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7081 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7141 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7201 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7261 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7321 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7381 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7441 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7501 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7561 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7621 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7681 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7741 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7801 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7861 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     7921 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctg
//
