LOCUS Exported 5715 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 Nsp1 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-FH-Nsp1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5715) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 5715) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5715 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 121..500 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 501..704 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 655..675 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 706..724 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 727..745 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 755..779 /label=LNCX /note="Human CMV promoter, forward primer" CDS 876..899 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 912..935 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(1553..1570) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1559..1783 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1916..1935) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2067..2114 /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 2122..3144 /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 3274..3395 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3311..3330) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3365..3384 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3444..3460) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3444..3460) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3457..3479) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3468..3484 /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(3492..3522) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3537..3558 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3675..3692) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3846..4434) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3926..3945) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4605..5465) /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 5228..5247 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5466..5570) /gene="bla" /label=AmpR promoter primer_bind complement(5645..5664) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga 61 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt 121 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 181 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 241 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 301 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 361 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 421 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 481 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 541 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 601 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 661 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata 721 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg 781 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc 841 tccggactct agcgtttaaa cttaagcttg agatggacta taaagatgac gatgacaagg 901 ccgcagccgc acatcaccac catcatcacc atcacggatc cgggatggaa tccttggtgc 961 cgggttttaa tgagaaaaca cacgtccagt tgtccctccc agttttgcag gtgcgagatg 1021 tgcttgtccg ggggttcggg gactccgtgg aggaggtact cagcgaagct agacagcatc 1081 tcaaggacgg aacttgcgga ctcgttgaag ttgaaaaggg ggtactgcca cagcttgagc 1141 agccctacgt cttcattaag cgatctgatg ccagaacggc tcctcatgga cacgtaatgg 1201 tggagttggt cgctgagctt gagggaatac agtacggcag atctggagag acgctgggcg 1261 tactcgtacc gcatgttggt gagatccctg ttgcatacag gaaagtattg ttgcgaaaaa 1321 acggtaataa aggagcgggt ggtcatagct acggggcgga tcttaaatct tttgaccttg 1381 gggatgaact gggcactgat ccgtacgaag attttcagga gaattggaat accaaacatt 1441 caagcggcgt gacgagggaa cttatgagag agctgaacgg cggggcctag gagatatcca 1501 gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact 1561 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 1621 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 1681 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 1741 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 1801 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 1861 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 1921 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 1981 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 2041 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa 2101 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt 2161 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct 2221 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc 2281 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt 2341 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt 2401 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg 2461 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc 2521 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt 2581 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc 2641 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc 2701 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc 2761 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc 2821 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg 2881 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc 2941 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac 3001 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg 3061 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc 3121 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc 3181 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 3241 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 3301 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 3361 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 3421 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 3481 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 3541 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 3601 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 3661 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 3721 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 3781 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3841 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3901 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3961 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4021 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4081 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4141 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4201 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4261 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4321 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4381 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4441 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4501 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4561 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4621 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 4681 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 4741 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 4801 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 4861 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 4921 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 4981 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 5041 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 5101 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 5161 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 5221 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 5281 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 5341 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 5401 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 5461 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 5521 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 5581 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 5641 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 5701 tggaggtcgc tgagt //