LOCUS Exported 6216 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 Nsp15 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-FH-Nsp15 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6216) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6216) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6216 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 820..838 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 869..893 /label=LNCX /note="Human CMV promoter, forward primer" CDS 990..1013 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 1026..1049 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(2168..2185) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2174..2398 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2531..2550) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2682..2729 /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 2737..3759 /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 3889..4010 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3926..3945) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3980..3999 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4059..4075) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4059..4075) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4072..4094) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4083..4099 /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(4107..4137) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4152..4173 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4290..4307) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4461..5049) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4541..4560) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5220..6080) /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 5843..5862 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6081..6185) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttgagatgg actataaaga tgacgatgac aaggccgcag 1021 ccgcacatca ccaccatcat caccatcacg gatccgggat gagtctggaa aacgtcgctt 1081 ttaatgtcgt aaataagggg cattttgatg gtcaacaggg ggaagttcct gtttccataa 1141 tcaacaacac tgtgtatact aaagtagatg gtgtggatgt cgaacttttt gaaaataaga 1201 cgaccctgcc ggtaaacgtg gcgttcgaat tgtgggcgaa gaggaatata aaaccagtac 1261 cggaagtaaa gatcttgaac aatcttggtg tggatatagc ggccaatacc gtgatatggg 1321 attataaaag ggatgcacct gctcacatca gtactattgg tgtctgctcc atgacggaca 1381 tcgctaaaaa gcctaccgag acgatctgcg ctccgctgac tgtgttcttt gatggaaggg 1441 tcgatgggca agtggacctg tttcggaacg caaggaacgg ggttctcata actgaagggt 1501 ccgtcaaggg gcttcagcca tcagtaggac ctaaacaggc ttcactcaat ggcgtgacgc 1561 ttattggcga ggcggtgaag actcagttta actactataa aaaagtagat ggcgtagtac 1621 aacaactgcc cgaaacgtat tttacgcagt cacgcaacct gcaagagttc aagccacgct 1681 cacagatgga gatcgacttc cttgagcttg cgatggacga gttcattgag agatacaagc 1741 tcgaagggta tgcgtttgaa catatagtat atggagactt ctcccatagt cagctgggtg 1801 ggcttcacct gctcatcggg ctggcgaagc gattcaaaga atctcccttc gaactcgaag 1861 actttatccc aatggactcc acggtgaaga actactttat tacagacgcc caaactggta 1921 gttctaaatg cgtgtgctca gttatagacc tgctgcttga tgactttgta gaaataataa 1981 aatcacagga cctttcagtc gtgtccaagg tggttaaggt cactatagac tatacagaaa 2041 tatctttcat gctgtggtgc aaggacggac acgtggaaac tttctaccct aagttgcaag 2101 cctaggagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc 2161 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 2221 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 2281 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 2341 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct 2401 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc 2461 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 2521 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 2581 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 2641 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt 2701 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg 2761 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg 2821 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg 2881 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg 2941 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat 3001 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc 3061 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag 3121 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat 3181 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc 3241 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc 3301 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc 3361 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc 3421 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc 3481 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt 3541 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg 3601 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc 3661 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga 3721 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat 3781 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg 3841 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt 3901 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 3961 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt 4021 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga 4081 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 4141 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 4201 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 4261 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 4321 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 4381 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 4441 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 4501 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 4561 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 4621 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 4681 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 4741 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 4801 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 4861 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 4921 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 4981 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 5041 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 5101 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 5161 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 5221 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 5281 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 5341 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 5401 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 5461 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 5521 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 5581 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 5641 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 5701 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 5761 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 5821 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 5881 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 5941 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6001 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6061 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 6121 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 6181 ccgcgcacat ttccccgaaa agtgccacct gacgtc //