LOCUS Exported 5595 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 Nsp10 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-FH-Nsp10 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5595) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 5595) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5595 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 96..475 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 476..679 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 630..650 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 681..699 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 702..720 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 730..754 /label=LNCX /note="Human CMV promoter, forward primer" CDS 851..874 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 887..910 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(1408..1425) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1414..1638 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1771..1790) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 1922..1969 /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 1977..2999 /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 3129..3250 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3166..3185) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3220..3239 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3299..3315) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3299..3315) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3312..3334) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3323..3339 /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(3347..3377) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3392..3413 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3530..3547) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3701..4289) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3781..3800) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4460..5320) /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 5083..5102 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5321..5425) /gene="bla" /label=AmpR promoter primer_bind complement(5500..5519) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc 61 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 421 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 481 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 601 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 661 ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga 721 tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt 781 tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa 841 gcttgagatg gactataaag atgacgatga caaggccgca gccgcacatc accaccatca 901 tcaccatcac ggatccggga tggctggcaa cgcaacggag gttcctgcaa atagtacagt 961 tctcagcttt tgcgcttttg cagttgacgc cgctaaggca tataaggatt atttggcatc 1021 cgggggtcaa ccaatcacca actgcgtcaa aatgctctgc actcatacag gaaccggaca 1081 ggcaatcact gtgactcctg aggcaaacat ggatcaggag agcttcggag gggccagttg 1141 ctgtttgtac tgtagatgcc atatagatca tcctaacccc aaagggttct gcgatcttaa 1201 aggtaaatat gtacaaatac ccacaacttg cgcgaacgac cccgttggtt ttacccttaa 1261 aaatactgta tgcaccgtct gcgggatgtg gaagggttac ggctgctcat gtgaccaact 1321 cagagagccc atgctgcagg cctaggagat atccagcaca gtggcggccg ctcgagtcta 1381 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 1441 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 1501 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 1561 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 1621 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc 1681 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 1741 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 1801 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 1861 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct 1921 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa 1981 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct 2041 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag 2101 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg 2161 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat 2221 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc 2281 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg 2341 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc 2401 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc 2461 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc 2521 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca 2581 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg 2641 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg 2701 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc 2761 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt 2821 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga 2881 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag 2941 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc 3001 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg 3061 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg 3121 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 3181 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 3241 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt 3301 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 3361 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 3421 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 3481 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 3541 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 3601 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 3661 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 3721 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 3781 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 3841 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 3901 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 3961 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4021 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 4081 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 4141 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 4201 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4261 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4321 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4381 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4441 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 4501 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 4561 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 4621 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 4681 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 4741 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 4801 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 4861 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 4921 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 4981 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 5041 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 5101 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5161 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5221 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5281 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5341 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5401 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg 5461 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc 5521 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag 5581 caaaatttaa gctac //