LOCUS Exported 6381 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression of untagged SARS-CoV-2 N protein under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-Nprot SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6381) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6381) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6381 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 112..491 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 492..695 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 646..666 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 697..715 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 718..736 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 746..770 /label=LNCX /note="Human CMV promoter, forward primer" primer_bind complement(2210..2227) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2216..2440 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2573..2592) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2724..2771 /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 2779..3801 /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 3931..4052 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3968..3987) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4022..4041 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4101..4117) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4101..4117) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4114..4136) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4125..4141 /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(4149..4179) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4194..4215 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4332..4349) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4503..5091) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4583..4602) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5262..6122) /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 5885..5904 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6123..6227) /gene="bla" /label=AmpR promoter primer_bind complement(6302..6321) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta 61 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat 121 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 181 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 241 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 301 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 361 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 421 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 481 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 541 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 601 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 661 ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat agagatctcc 721 ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc gcctggagac 781 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc 841 tagcgtttaa acttaagctt ggtaccgagc tcggatccgg gatgtccgac aatggtccgc 901 agaaccagag aaacgcaccc aggataacat ttgggggtcc aagtgacagt acaggatcta 961 accagaatgg ggagcgaagc ggggcacggt ctaagcaacg cagaccccag ggtcttccta 1021 ataatacagc atcatggttt accgctctca cacaacacgg aaaagaagac ttgaagttcc 1081 ctcgcgggca gggggtcccg atcaatacga atagttctcc cgatgatcaa atcggttatt 1141 accgaagggc cacccgcaga attcgaggag gggatggtaa gatgaaggat cttagtccaa 1201 ggtggtattt ctattacctc ggcacgggcc cggaggcggg cctcccatac ggggctaaca 1261 aggacggaat catctgggtg gcgaccgagg gtgccctcaa cactcccaag gatcatatag 1321 gaacgcggaa tccagcaaat aatgcggcta tagtgttgca acttccgcaa ggaacaaccc 1381 tccctaaggg attctatgca gaggggtcca ggggaggttc tcaagctagt tctcgctcat 1441 caagccgatc tcggaattca tctcggaatt caacgccagg atctagtcga ggcacaagtc 1501 cggcccggat ggccggaaac ggcggcgatg ccgcgttggc actcctgctc ttggatcgcc 1561 ttaaccaact tgaatctaaa atgagtggta aggggcagca acaacagggg cagacagtga 1621 caaaaaagag tgcagcagag gcgagtaaga agcccaggca aaaacgcaca gcaacgaaag 1681 catataacgt cacccaggca tttggtagac ggggccccga acagacacaa ggtaacttcg 1741 gagatcagga actcatccgg caaggtaccg actacaagca ttggccacag atcgcgcagt 1801 ttgctcccag tgcgtccgct ttcttcggaa tgtctcggat aggcatggag gttacgccga 1861 gtggcacgtg gcttacatat accggagcaa tcaaactgga tgacaaagat ccaaatttca 1921 aagatcaggt tatcttgttg aacaaacata ttgatgccta taaaacgttt ccacccacag 1981 aacctaaaaa agacaagaag aaaaaagccg acgaaacgca agcccttcca cagaggcaga 2041 aaaagcagca aaccgtcact ctgctgcctg ctgccgacct ggatgacttc tctaagcagc 2101 tccaacaatc aatgtcaagt gccgattcaa cccaggccta agcctaggag atatccagca 2161 cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 2221 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 2281 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 2341 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 2401 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 2461 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt 2521 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 2581 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 2641 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 2701 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt 2761 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg 2821 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt 2881 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat 2941 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg 3001 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca 3061 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc 3121 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca 3181 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct 3241 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg 3301 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg 3361 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt 3421 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg 3481 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag 3541 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat 3601 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca 3661 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc 3721 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact 3781 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat 3841 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 3901 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 3961 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4021 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4081 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4141 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 4201 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 4261 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 4321 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 4381 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 4441 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4501 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4561 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4621 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4681 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4741 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4801 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4861 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4921 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 4981 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5041 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5101 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5161 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 5221 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 5281 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5341 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5401 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5461 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5521 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 5581 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5641 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 5701 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5761 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5821 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 5881 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5941 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6001 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6061 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6121 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 6181 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 6241 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca 6301 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg 6361 aggtcgctga gtagtgcgcg a //