LOCUS Exported 7214 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4133406 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7214) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7214) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7214 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 596..616 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 620..685 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 754..875 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(765..784) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 819..838 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 990..1036 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1066..1202 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1247..1318 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1544..2132) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1624..1643) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2632..3841 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3678..3697 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3845..4873 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" polyA_signal 4900..4948 /note="synthetic polyadenylation signal" CDS 5214..6074 /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 complement(5432..5451) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6087..6223 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6368..6439 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6444..6490 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6508..6556 /note="synthetic polyadenylation signal" enhancer 6637..6940 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6942..7144 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7095..7115 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgata gcctcagagt ttagttccct tccatcatat gcagcttttg ctactgctca 61 agaagcttat gagcaggctg ttgctaatgg tgattctgaa gttgttctta aaaagttgaa 121 gaagtctttg aatgtggcta aatctgaatt tgaccgtgat gcagccatgc aacgtaagtt 181 ggaaaagatg gctgatcaag ctatgaccca aatgtataaa caggctagat ctgaggacaa 241 gagggcaaaa gttactagtg ctatgcagac aatgcttttc actatgctta gaaagttgga 301 taatgatgca ctcaacaaca ttatcaacaa tgcaagagat ggttgtgttc ccttgaacat 361 aatacctctt acaacagcag ccaaactaat ggttgtcata ccagactata acacatataa 421 aaatacgtgt gatggtacaa catttactta tgcatcagca ttgtgggaaa tccaacaggt 481 tgtagatgca gatagtaaaa ttgttcaact tagtgaaatt agtatggaca attcacctaa 541 tttagcatgg cctcttattg taacagcttt aagggccaat tctgctgtca aattagaaaa 601 cctttacttt cagtcgatgg attataagga ccacgatgga gactacaaag atcatgacat 661 tgattacaag gacgacgatg acaaataagg atccgcaggc ctctgctagc ttgactgact 721 gagatacagc gtaccttcag ctcacagaca tgataagata cattgatgag tttggacaaa 781 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt 841 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta 901 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat 961 gtggtattgg cccatctcta tcggtatcgt agcataaccc cttggggcct ctaaacgggt 1021 cttgaggggt tttttgtgcc cctcgggccg gattgctatc taccggcatt ggcgcagaaa 1081 aaaatgcctg atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg 1141 atacggcttc cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt 1201 aaggtcgtca gctatcctgc aggcgatctc tcgatttcga tcaagacatt cctttaatgg 1261 tcttttctgg acaccactag gggtcagaag tagttcatca aactttcttc cctccctaat 1321 ctcattggtt accttgggct atcgaaactt aattaaccag tcaagtcagc tacttggcga 1381 gatcgacttg tctgggtttc gactacgctc agaattgcgt cagtcaagtt cgatctggtc 1441 cttgctattg cacccgttct ccgattacga gtttcattta aatcatgtga gcaaaaggcc 1501 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 1561 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 1621 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 1681 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 1741 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 1801 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 1861 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 1921 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 1981 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 2041 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 2101 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 2161 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 2221 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 2281 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 2341 tctgtctatt tcgttcatcc atagttgcat ttaaatttcc gaactctcca aggccctcgt 2401 cggaaaatct tcaaaccttt cgtccgatcc atcttgcagg ctacctctcg aacgaactat 2461 cgcaagtctc ttggccggcc ttgcgccttg gctattgctt ggcagcgcct atcgccaggt 2521 attactccaa tcccgaatat ccgagatcgg gatcacccga gagaagttca acctacatcc 2581 tcaatcccga tctatccgag atccgaggaa tatcgaaatc ggggcgcgcc tggcctccgc 2641 gccgggtttt ggcgcctccc gcgggcgccc ccctcgtcac ggcgagcgct gccacgtcag 2701 acgaagggcg caggagcgtc ctgatccttc cgcccggacg ctcaggacag cggcccgctg 2761 ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag gacgggactt 2821 gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg aaaagtagtc 2881 ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat gattatataa 2941 ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt cgcggttctt 3001 gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct ggccggggct 3061 ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc caagggctgt 3121 agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg cagcaaaatg 3181 gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa 3241 acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgagccctt cgctaatgcg 3301 ggaaagctct tattcgggtg agatgggctg ggcaccatct ggggaccctg acgtgaagtt 3361 tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt atggcggtgc 3421 cgttgggcag tgcacccgta cctttgggag cgcgcgccct cgtcgtgtcg tgacgtcacc 3481 cgttctgttg gcttataatg cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt 3541 tctccgtcgc aggacgcagg gttcgggcct agggtaggct ctcctgaatc gacaggcgcc 3601 ggacctctgg tgaggggagg gataagtgag gcgtcagttt ctttggtcgg ttttatgtac 3661 ctatcttctt aagtagctga agctccggtt ttgaactatg cgctcggggt tggcgagtgt 3721 gttttgtgaa gttttttagg caccttttga aatgtaatca tttgggtcaa tatgtaattt 3781 tcagtgttag acttgtaaat tgtccgctaa attctggccg tttttggctt ttttgttaga 3841 caacatgggt aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa 3901 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag 3961 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta 4021 caaagatcgt tatgtttatc ggcactttgc atccgccgcg ctcccgattc cggaagtgct 4081 tgacattggg gagttcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt 4141 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc 4201 aatggatgcc atcgctgccg ccgatcttag ccagacgagc gggttcggcc cattcggacc 4261 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca 4321 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 4381 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 4441 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag 4501 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg 4561 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg 4621 atcgccccgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt 4681 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 4741 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgccg ccgtctggac 4801 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag 4861 ggcaaaggaa taagctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga 4921 agatggatgt gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa 4981 cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga 5041 atccagcttg ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc 5101 agcgtaccga tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag 5161 tcctagcttt tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta 5221 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 5281 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg 5341 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 5401 gctttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 5461 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 5521 attcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 5581 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac 5641 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 5701 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 5761 caatggcaac aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc 5821 aacagttgat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 5881 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 5941 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 6001 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 6061 ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc 6121 tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg 6181 cccacttcca tacgtgtcct ccttaccaga aatttatcct taagatcgtt taaactcgac 6241 tctggctcta tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg 6301 ctcgtcgggc atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg 6361 ctcccgacat cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca 6421 gcagcttcag ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg 6481 ggttatgcta tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat 6541 ggatagcgat tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac 6601 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 6661 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 6721 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 6781 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 6841 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 6901 ttggcagtac atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta 6961 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 7021 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 7081 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 7141 agctggttta gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac 7201 ccgccagcgg ccgc //