LOCUS Exported 6981 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 Nsp13 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-FH-Nsp13 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6981) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6981) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6981 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 151..530 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 531..734 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 685..705 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 736..754 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 757..775 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 785..809 /label=LNCX /note="Human CMV promoter, forward primer" CDS 906..929 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 942..965 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(2849..2866) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2855..3079 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3212..3231) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3363..3410 /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 3418..4440 /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 4570..4691 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4607..4626) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4661..4680 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4740..4756) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4740..4756) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4753..4775) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4764..4780 /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(4788..4818) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4833..4854 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4971..4988) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5142..5730) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5222..5241) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5901..6761) /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 6524..6543 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6762..6866) /gene="bla" /label=AmpR promoter primer_bind complement(6941..6960) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa 61 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc 121 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 541 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 601 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 661 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 721 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc 781 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 841 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttg 901 agatggacta taaagatgac gatgacaagg ccgcagccgc acatcaccac catcatcacc 961 atcacggatc cgggatggct gttggagcct gtgtgttgtg taacagccag acttccctga 1021 gatgcggggc gtgcatacgc aggccgtttc tgtgctgtaa gtgttgttac gatcacgtca 1081 tttctacaag ccacaagctg gtcttgagcg ttaaccctta tgtttgtaat gcccctggct 1141 gcgatgttac tgatgtcacc cagttgtatt tgggtgggat gtcatattac tgtaagagcc 1201 ataagccacc gatctcattc cccttgtgcg cgaacggaca agtatttggt ttgtacaaaa 1261 acacgtgtgt aggatcagat aacgtaaccg atttcaatgc tattgcgacg tgtgattgga 1321 caaatgcagg agattacatt cttgccaata cttgcacaga aagacttaaa ttgttcgctg 1381 cggaaacatt gaaagcaact gaagagacgt ttaagctgtc atacggcatt gccaccgtga 1441 gggaggtgct gtctgaccga gagctccatc tgtcctggga ggtcgggaaa ccccgacctc 1501 cgttgaacag aaactacgtg tttaccggtt accgcgtcac aaagaattca aaggtgcaga 1561 taggagaata tactttcgaa aagggagatt acggagacgc ggtggtttat cgaggcacga 1621 cgacctacaa gctgaatgta ggcgattatt ttgtccttac atctcatacg gtcatgccat 1681 tgagcgctcc cactctggtc ccacaggaac attatgtgag gattacaggc ctgtatccca 1741 ctttgaacat aagtgatgaa ttttcctcta atgtcgcaaa ttaccaaaaa gtaggtatgc 1801 agaagtattc cactctccag gggccgcccg gaactgggaa gagtcacttc gcaattgggt 1861 tggctctgta ttatccaagc gcacgaatag tttacacagc atgtagccat gcggccgttg 1921 atgcgctgtg cgagaaagcg ctgaaatacc ttcctattga caaatgcagc cggatcatcc 1981 ccgcacgcgc ccgcgtggag tgttttgata agttcaaagt aaattcaacc ctggagcagt 2041 atgtgttttg tacggtgaat gccctgccag aaacaactgc ggacatagtg gtgtttgacg 2101 aaatatcaat ggcgactaat tatgacctta gtgtggtgaa cgctagattg cgagccaagc 2161 actacgtata tataggcgat ccagctcaac tccccgctcc tcggacgctc ctgaccaagg 2221 gaaccttgga gccagaatac ttcaattccg tctgccgact gatgaagacc ataggcccag 2281 acatgttctt ggggacgtgt cgccgctgtc cagcagaaat agtggacacg gttagcgcgc 2341 ttgtgtacga caataaactc aaggcacaca aagataagtc cgcccagtgt tttaaaatgt 2401 tctataaagg tgttataact catgatgtct cctctgcaat caatcgaccc cagatcgggg 2461 ttgtccgaga gtttctgaca cggaatcccg cttggcgaaa ggcggtgttt atctctccat 2521 acaactctca gaatgccgtg gcctctaaga tccttggtct gcccacgcaa accgtagatt 2581 ctagccaggg tagcgagtat gactacgtta tttttactca gacaacagaa actgctcata 2641 gttgcaacgt caataggttc aatgtggcca ttacaagggc gaaggtgggt attctgtgta 2701 ttatgagtga tcgagacctg tacgataaac ttcagtttac ctcactggag atcccccgca 2761 gaaatgtggc caccctgcaa gcctaggaga tatccagcac agtggcggcc gctcgagtct 2821 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 2881 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 2941 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 3001 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 3061 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 3121 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 3181 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 3241 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 3301 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc 3361 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa 3421 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc 3481 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga 3541 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat 3601 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa 3661 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac 3721 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc 3781 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt 3841 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg 3901 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg 3961 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac 4021 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc 4081 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg 4141 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc 4201 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat 4261 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg 4321 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta 4381 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag 4441 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc 4501 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc 4561 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 4621 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 4681 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg 4741 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 4801 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 4861 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 4921 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 4981 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 5041 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5101 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 5161 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 5221 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5281 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5341 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5401 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5461 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 5521 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 5581 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 5641 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 5701 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 5761 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 5821 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 5881 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 5941 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 6001 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 6061 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 6121 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 6181 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 6241 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 6301 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 6361 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 6421 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 6481 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 6541 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 6601 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 6661 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 6721 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 6781 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 6841 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac 6901 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg 6961 catagttaag ccagtatctg c //