LOCUS Exported 6003 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression of untagged SARS-CoV-2 Nsp7-Nsp8 fusion protein under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA-FRT-TO-Nsp7-Nsp8_GC3opt SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6003) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6003) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6003 /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" primer_bind complement(1955..1972) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1961..2185 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2318..2337) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2469..2516 /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 2524..3546 /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 3676..3797 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3713..3732) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3767..3786 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3846..3862) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3846..3862) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3859..3881) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3870..3886 /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(3894..3924) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3939..3960 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4077..4094) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4248..4836) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4328..4347) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5007..5867) /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 5630..5649 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5868..5972) /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 cttggtaccg agctcggatc cgggatgagc aagatgagcg 1021 acgtgaagtg caccagcgtg gtgctgctgt ctgttctgca gcagctgaga gtggaaagca 1081 gcagcaaact gtgggcccag tgtgtccagc tgcacaacga tattctgctg gccaaggaca 1141 ccaccgaggc cttcgagaag atggtgtctc tgctgagcgt gctgctgagc atgcaaggcg 1201 ccgtggacat caacaagctg tgcgaggaaa tgctggacaa ccgggccaca ctgcagggct 1261 ctgctggatc tgctgctggc tctggcgaat ttgccattgc cagcgagttc agcagcctgc 1321 ctagctatgc cgcctttgcc acagctcaag aggcctatga acaggccgtg gccaatggcg 1381 attctgaggt ggtgctgaag aagctgaaaa agtccctgaa cgtggccaag agcgagttcg 1441 acagagatgc cgccatgcag cggaagctgg aaaagatggc cgaccaggcc atgacacaga 1501 tgtacaagca ggccagaagc gaggacaaga gggccaaagt gacctctgcc atgcagacca 1561 tgctgttcac catgctgaga aagctggata acgacgccct gaacaacatc atcaacaacg 1621 ccagagatgg ctgcgtgccc ctgaatatca tccctctgac aaccgccgcc aagctgatgg 1681 tggtcatccc cgactacaac acctacaaga atacctgcga cggcaccacc ttcacctacg 1741 cctctgctct gtgggagatt cagcaggtcg tggacgccga cagcaagatc gtgcagctga 1801 gcgagatcag catggacaac agccccaatc tggcctggcc tctgattgtg acagccctga 1861 gagccaacag cgccgtgaaa ctgcaggcct aggagatatc cagcacagtg gcggccgctc 1921 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 1981 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2041 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2101 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 2161 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg 2221 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 2281 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 2341 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 2401 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 2461 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg 2521 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 2581 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 2641 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 2701 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 2761 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 2821 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat 2881 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 2941 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 3001 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 3061 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 3121 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 3181 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 3241 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg 3301 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 3361 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 3421 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 3481 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 3541 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 3601 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 3661 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 3721 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 3781 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 3841 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 3901 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 3961 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 4021 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4081 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4141 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4201 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4261 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4321 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4381 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4441 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4501 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4561 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4621 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4681 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 4741 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 4801 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 4861 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 4921 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 4981 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5041 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5101 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5161 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5221 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5281 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5341 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 5401 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 5461 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 5521 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 5581 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 5641 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 5701 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 5761 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 5821 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 5881 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 5941 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 6001 gtc //