LOCUS Exported 6705 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression of untagged SARS-CoV-2 Nsp14 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-Nsp14 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6705) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6705) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6705 /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" primer_bind complement(2573..2590) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2579..2803 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2936..2955) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3087..3134 /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 3142..4164 /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 4294..4415 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4331..4350) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4385..4404 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4464..4480) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4464..4480) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4477..4499) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4488..4504 /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(4512..4542) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4557..4578 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4695..4712) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4866..5454) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4946..4965) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5625..6485) /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 6248..6267 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6486..6590) /gene="bla" /label=AmpR promoter primer_bind complement(6665..6684) /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 gtaccgagct cggatccggg atggctgaaa atgtgactgg tctcttcaaa gattgctcca 961 aggtgattac gggcctgcat cccacccagg cccccacaca tctttcagta gacacgaaat 1021 ttaaaactga aggtctttgt gttgacatcc ctggcattcc gaaggatatg acttatcgcc 1081 ggcttatctc catgatgggg tttaagatga attaccaggt caacgggtac ccaaacatgt 1141 tcatcacacg agaggaggca attcggcacg ttagagcatg gattggtttt gacgttgagg 1201 gatgccacgc cactcgagag gcggtcggga ccaacttgcc actccagttg ggcttctcca 1261 ccggcgtcaa tctggtcgca gtacccacag gttacgtgga cacgcccaat aacacagact 1321 tttcaagagt aagcgctaaa cccccacccg gagatcagtt caaacacttg atacccctga 1381 tgtacaaagg tcttccctgg aacgttgtcc gaatcaaaat cgtccagatg ctctctgata 1441 cgctcaagaa cctgagcgat cgagtcgttt tcgtactctg ggcgcatggt ttcgagctga 1501 cctccatgaa gtacttcgta aaaatagggc ctgagagaac atgctgtctg tgtgatcgaa 1561 gagctacctg ttttagcacc gcttccgata catatgcttg ctggcatcat tcaataggtt 1621 tcgattacgt atacaaccca ttcatgatcg atgtgcagca gtgggggttc actgggaacc 1681 tgcaatcaaa ccacgatctg tattgtcaag tacacggtaa tgcgcacgtc gcttcatgtg 1741 atgccatcat gacgcggtgc ctggcagtac atgagtgctt tgtaaagagg gttgattgga 1801 cgatagagta tcctattatc ggagacgaac tcaaaataaa tgctgcctgc cgcaaagttc 1861 agcatatggt tgtaaaagcg gcccttctcg cggataaatt tccggttttg catgacatcg 1921 gaaatccgaa agcaataaag tgcgtcccgc aggcagacgt ggaatggaaa ttttatgatg 1981 cacaaccttg cagtgataag gcctataaaa ttgaagaact gttctacagc tacgccaccc 2041 actctgataa gttcacagac ggagtctgtc ttttttggaa ttgtaacgtc gatcggtatc 2101 cagctaattc tattgtctgt cgatttgaca cgagggtttt gtcaaacctg aaccttccag 2161 gatgtgatgg aggttctttg tacgtcaata aacacgcatt ccacacgccc gcctttgata 2221 aatctgcatt cgtaaatctc aagcagcttc ccttctttta ctacagtgat tccccatgtg 2281 aatcacatgg caagcaagtt gtcagcgata tagactatgt gccccttaag tctgcaacct 2341 gcatcacgcg ctgtaatttg ggaggtgcag tctgtcggca tcatgcgaat gagtataggc 2401 tttatctcga cgcgtacaac atgatgattt cagcggggtt ttcattgtgg gtatataagc 2461 aattcgacac ttataacttg tggaacacat ttacgcgcct ccaggcctag gagatatcca 2521 gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact 2581 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 2641 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 2701 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 2761 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 2821 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 2881 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 2941 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 3001 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 3061 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa 3121 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt 3181 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct 3241 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc 3301 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt 3361 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt 3421 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg 3481 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc 3541 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt 3601 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc 3661 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc 3721 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc 3781 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc 3841 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg 3901 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc 3961 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac 4021 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg 4081 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc 4141 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc 4201 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 4261 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 4321 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 4381 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 4441 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4501 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4561 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4621 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4681 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4741 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4801 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4861 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4921 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4981 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 5041 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 5101 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 5161 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 5221 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5281 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 5341 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5401 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5461 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5521 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5581 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5641 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 5701 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 5761 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 5821 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 5881 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 5941 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6001 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 6061 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 6121 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6181 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6241 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6301 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6361 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6421 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6481 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6541 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6601 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 6661 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgc //