LOCUS Exported 7974 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 Nsp12 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-FH-Nsp12 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7974) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 7974) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7974 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /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(5754..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(6867..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 //