LOCUS Exported 5361 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 Orf6 under control of a tetracycline-inducible promoter. ACCESSION . VERSION . KEYWORDS pcDNA5-FRT-TO-FH-Orf6 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5361) TITLE Tollervey SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 5361) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5361 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 69..448 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 449..652 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 603..623 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 654..672 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 675..693 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 703..727 /label=LNCX /note="Human CMV promoter, forward primer" CDS 824..847 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 860..883 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(1147..1164) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1153..1377 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1510..1529) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 1661..1708 /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 1716..2738 /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 2868..2989 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2905..2924) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2959..2978 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3038..3054) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3038..3054) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3051..3073) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3062..3078 /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(3086..3116) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3131..3152 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3269..3286) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3440..4028) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3520..3539) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4199..5059) /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 4822..4841 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5060..5164) /gene="bla" /label=AmpR promoter primer_bind complement(5239..5258) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata 61 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 121 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 181 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 241 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 301 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 361 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 421 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 481 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 541 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 601 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat 661 cagtgataga gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg 721 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg 781 atccagcctc cggactctag cgtttaaact taagcttgag atggactata aagatgacga 841 tgacaaggcc gcagccgcac atcaccacca tcatcaccat cacggatccg ggatgtttca 901 tttggtggac tttcaggtta caatcgcgga gatcctgctc ataataatga ggacctttaa 961 ggtgtcaata tggaaccttg attacattat caacctgatt attaagaacc tcagcaaaag 1021 cctgacagaa aacaaataca gccagttgga tgaagaacaa cctatggaaa tcgactaagc 1081 ctaggagata tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct 1141 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 1201 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 1261 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 1321 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 1381 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 1441 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 1501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 1561 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 1621 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt 1681 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt 1741 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg 1801 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg 1861 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg 1921 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt 1981 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg 2041 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga 2101 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt 2161 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg 2221 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg 2281 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc 2341 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg 2401 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg 2461 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg 2521 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc 2581 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg 2641 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa 2701 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt 2761 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 2821 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 2881 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 2941 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 3001 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 3061 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 3121 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 3181 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 3241 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 3301 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 3361 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 3421 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 3481 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3541 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 3601 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 3661 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 3721 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 3781 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 3841 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 3901 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 3961 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 4021 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 4081 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 4141 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 4201 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 4261 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 4321 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 4381 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 4441 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 4501 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 4561 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 4621 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 4681 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 4741 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 4801 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 4861 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 4921 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 4981 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 5041 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 5101 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 5161 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc 5221 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct 5281 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag 5341 gcaaggcttg accgacaatt g //