LOCUS Exported 6971 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 Nsp13 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-Nsp13-HF_GC3opt SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6971) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6971) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6971 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 132..511 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 512..715 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 666..686 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 717..735 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 738..756 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 766..790 /label=LNCX /note="Human CMV promoter, forward primer" CDS 2720..2743 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" CDS 2756..2779 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" primer_bind complement(2820..2837) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2826..3050 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3183..3202) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3334..3381 /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 3389..4411 /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 4541..4662 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4578..4597) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4632..4651 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4711..4727) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4711..4727) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4724..4746) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4735..4751 /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(4759..4789) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4804..4825 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4942..4959) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5113..5701) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5193..5212) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5872..6732) /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 6495..6514 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6733..6837) /gene="bla" /label=AmpR promoter primer_bind complement(6912..6931) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa 61 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag 121 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 181 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 241 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 301 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 361 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 421 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 481 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 541 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 601 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 661 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc 721 tatcagtgat agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa 781 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 841 ccgatccagc ctccggactc tagcgtttaa acttaagctt ggtaccgagc tcggatccgg 901 gatggctgtg ggagcttgcg tgctgtgtaa tagccagacc agcctgagat gcggcgcctg 961 tatcagacgg cctttcctgt gctgcaagtg ctgctacgac cacgtgatca gcaccagcca 1021 caaactggtg ctgtccgtga atccctacgt gtgcaatgcc cctggctgcg acgtgaccga 1081 tgtgacacag ctgtatctcg gcggcatgag ctactactgc aagagccaca agcctccaat 1141 cagcttccct ctgtgcgcca acggacaggt gttcggcctg tacaagaata cctgcgtggg 1201 cagcgacaat gtgaccgact tcaacgccat tgccacctgt gactggacca acgccggcga 1261 ttacatcctg gccaatacct gcaccgagcg gctgaagctg tttgccgccg aaacactgaa 1321 ggccaccgag gaaaccttca agctgagcta cggaatcgcc accgtgcggg aagtgctgag 1381 cgatagagaa ctgcacctga gctgggaagt gggcaagcct agacctcctc tgaaccggaa 1441 ctacgtgttc accggctaca gagtgaccaa gaacagcaag gtgcagatcg gcgagtacac 1501 cttcgagaag ggcgattatg gcgacgccgt ggtgtacaga ggcaccacca cctacaagct 1561 gaacgtgggc gactacttcg tgctgacaag ccacaccgtg atgcctctgt ctgcccctac 1621 actggtgccc caagaacact acgtgcggat caccggactg taccccacac tgaacatcag 1681 cgacgagttc agcagcaacg tggccaacta ccagaaagtg ggcatgcaga agtacagcac 1741 actgcagggc ccacctggca caggcaaaag ccactttgcc attggactgg ccctgtacta 1801 ccccagcgcc agaatcgtgt acaccgcctg ttctcacgcc gccgttgatg ccctgtgtga 1861 aaaggccctg aagtacctgc ctatcgacaa gtgcagccgg atcatccctg ccagagccag 1921 agtggaatgc ttcgacaagt tcaaagtgaa cagcaccctg gaacagtacg tgttctgcac 1981 cgtgaacgcc ctgcctgaga caacagccga catcgtggtg ttcgacgaga tctccatggc 2041 caccaactac gacctgagcg tggtcaacgc cagactgaga gccaagcact acgtgtacat 2101 cggcgaccct gctcagctgc ccgctcctag aacactgctg acaaagggca cactggaacc 2161 cgagtacttc aacagcgtgt gccggctgat gaagacaatc ggccccgaca tgttcctggg 2221 cacctgtaga agatgccctg ccgagatcgt ggacaccgtg tctgccctgg tgtacgacaa 2281 caaactgaaa gcccacaagg acaagagcgc ccagtgcttc aagatgttct acaagggcgt 2341 gatcacccac gacgtgtcca gcgccatcaa cagacctcag atcggagttg tgcgcgagtt 2401 cctgaccaga aatcctgcct ggcggaaggc cgtgtttatc agcccctaca acagccagaa 2461 tgccgtggcc agcaagatcc tgggactgcc tacacagacc gtggatagct ctcagggcag 2521 cgagtacgat tacgtgatct tcacccagac caccgagaca gcccacagct gcaacgtgaa 2581 cagattcaat gtggccatca ccagggccaa agtgggaatc ctgtgcatca tgagcgaccg 2641 ggacctgtac gataagctgc agttcaccag cctggaaatc cccagacgga acgtggcaac 2701 actgcaggca tcgggcggac atcaccacca tcatcaccat cacgccgcag ccgcagatta 2761 taaagatgac gatgacaagt agctcgagtc tagagggccc gtttaaaccc gctgatcagc 2821 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2881 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2941 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 3001 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3061 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag 3121 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 3181 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 3241 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 3301 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc 3361 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga 3421 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga 3481 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag 3541 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct 3601 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc 3661 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct 3721 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg 3781 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg 3841 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc 3901 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg 3961 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac 4021 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat 4081 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag 4141 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga 4201 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg 4261 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag 4321 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg 4381 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc 4441 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 4501 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 4561 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 4621 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 4681 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 4741 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 4801 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 4861 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 4921 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 4981 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 5041 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 5101 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 5161 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 5221 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 5281 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 5341 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 5401 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 5461 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 5521 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 5581 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 5641 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 5701 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 5761 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 5821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 5881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 5941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 6001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 6061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 6121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 6181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 6241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 6301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 6361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 6421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 6481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 6541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 6601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 6661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 6721 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 6781 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 6841 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg 6901 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct 6961 tgtgtgttgg a //