LOCUS Exported 9374 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA polymerase. ACCESSION . VERSION . KEYWORDS pGBW-m4133572 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9374) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 9374) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9374 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 2798..2818 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2819..2845 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 2914..3035 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2925..2944) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2979..2998 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 3150..3196 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 3226..3362 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3407..3478 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3704..4292) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3784..3803) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4792..6001 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5838..5857 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 6005..7033 /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 7060..7108 /note="synthetic polyadenylation signal" CDS 7374..8234 /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(7592..7611) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 8247..8383 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 8528..8599 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 8604..8650 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 8668..8716 /note="synthetic polyadenylation signal" enhancer 8797..9100 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 9102..9304 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 9255..9275 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 9373..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggct gatgcacaat cgtttttaaa ccgggtttgc ggtgtaagtg cagcccgtct 61 tacaccgtgc ggcacaggca ctagtactga tgtcgtatac agggcttttg acatctacaa 121 tgataaagta gctggttttg ctaaattcct aaaaactaat tgttgtcgct tccaagaaaa 181 ggacgaagat gacaatttaa ttgattctta ctttgtagtt aagagacaca ctttctctaa 241 ctaccaacat gaagaaacaa tttataattt acttaaggat tgtccagctg ttgctaaaca 301 tgacttcttt aagtttagaa tagacggtga catggtacca catatatcac gtcaacgtct 361 tactaaatac acaatggcag acctcgtcta tgctttaagg cattttgatg aaggtaattg 421 tgacacatta aaagaaatac ttgtcacata caattgttgt gatgatgatt atttcaataa 481 aaaggactgg tatgattttg tagaaaaccc agatatatta cgcgtatacg ccaacttagg 541 tgaacgtgta cgccaagctt tgttaaaaac agtacaattc tgtgatgcca tgcgaaatgc 601 tggtattgtt ggtgtactga cattagataa tcaagatctc aatggtaact ggtatgattt 661 cggtgatttc atacaaacca cgccaggtag tggagttcct gttgtagatt cttattattc 721 attgttaatg cctatattaa ccttgaccag ggctttaact gcagagtcac atgttgacac 781 tgacttaaca aagccttaca ttaagtggga tttgttaaaa tatgacttca cggaagagag 841 gttaaaactc tttgaccgtt attttaaata ttgggatcag acataccacc caaattgtgt 901 taactgtttg gatgacagat gcattctgca ttgtgcaaac tttaatgttt tattctctac 961 agtgttccca cctacaagtt ttggaccact agtgagaaaa atatttgttg atggtgttcc 1021 atttgtagtt tcaactggat accacttcag agagctaggt gttgtacata atcaggatgt 1081 aaacttacat agctctagac ttagttttaa ggaattactt gtgtatgctg ctgaccctgc 1141 tatgcacgct gcttctggta atctattact agataaacgc actacgtgct tttcagtagc 1201 tgcacttact aacaatgttg cttttcaaac tgtcaaaccc ggtaatttta acaaagactt 1261 ctatgacttt gctgtgtcta agggtttctt taaggaagga agttctgttg aattaaaaca 1321 cttcttcttt gctcaggatg gtaatgctgc tatcagcgat tatgactact atcgttataa 1381 tctaccaaca atgtgtgata tcagacaact actatttgta gttgaagttg ttgataagta 1441 ctttgattgt tacgatggtg gctgtattaa tgctaaccaa gtcatcgtca acaacctaga 1501 caaatcagct ggttttccat ttaataaatg gggtaaggct agactttatt atgattcaat 1561 gagttatgag gatcaagatg cacttttcgc atatacaaaa cgtaatgtca tccctactat 1621 aactcaaatg aatcttaagt atgccattag tgcaaagaat agagctcgca ccgtagctgg 1681 tgtctctatc tgtagtacta tgaccaatag acagtttcat caaaaattat tgaaatcaat 1741 agccgccact agaggagcta ctgtagtaat tggaacaagc aaattctatg gtggttggca 1801 caacatgtta aaaactgttt atagtgatgt agaaaaccct caccttatgg gttgggatta 1861 tcctaaatgt gatagagcca tgcctaacat gcttagaatt atggcctcac ttgttcttgc 1921 tcgcaaacat acaacgtgtt gtagcttgtc acaccgtttc tatagattag ctaatgagtg 1981 tgctcaagta ttgagtgaaa tggtcatgtg tggcggttca ctatatgtta aaccaggtgg 2041 aacctcatca ggagatgcca caactgctta tgctaatagt gtttttaaca tttgtcaagc 2101 tgtcacggcc aatgttaatg cacttttatc tactgatggt aacaaaattg ccgataagta 2161 tgtccgcaat ttacaacaca gactttatga gtgtctctat agaaatagag atgttgacac 2221 agactttgtg aatgagtttt acgcatattt gcgtaaacat ttctcaatga tgatactctc 2281 tgacgatgct gttgtgtgtt tcaatagcac ttatgcatct caaggtctag tggctagcat 2341 aaagaacttt aagtcagttc tttattatca aaacaatgtt tttatgtctg aagcaaaatg 2401 ttggactgag actgacctta ctaaaggacc tcatgaattt tgctctcaac atacaatgct 2461 agttaaacag ggtgatgatt atgtgtacct tccttaccca gatccatcaa gaatcctagg 2521 ggccggctgt tttgtagatg atatcgtaaa aacagatggt acacttatga ttgaacggtt 2581 cgtgtcttta gctatagatg cttacccact tactaaacat cctaatcagg agtatgctga 2641 tgtctttcat ttgtacttac aatacataag aaagctacat gatgagttaa caggacacat 2701 gttagacatg tattctgtta tgcttactaa tgataacact tcaaggtatt gggaacctga 2761 gttttatgag gctatgtaca caccgcatac agtcttagaa aacctttact ttcagtcgta 2821 cccctatgac gtaccagatt atgcgtaagg atccgcaggc ctctgctagc ttgactgact 2881 gagatacagc gtaccttcag ctcacagaca tgataagata cattgatgag tttggacaaa 2941 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt 3001 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta 3061 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat 3121 gtggtattgg cccatctcta tcggtatcgt agcataaccc cttggggcct ctaaacgggt 3181 cttgaggggt tttttgtgcc cctcgggccg gattgctatc taccggcatt ggcgcagaaa 3241 aaaatgcctg atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg 3301 atacggcttc cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt 3361 aaggtcgtca gctatcctgc aggcgatctc tcgatttcga tcaagacatt cctttaatgg 3421 tcttttctgg acaccactag gggtcagaag tagttcatca aactttcttc cctccctaat 3481 ctcattggtt accttgggct atcgaaactt aattaaccag tcaagtcagc tacttggcga 3541 gatcgacttg tctgggtttc gactacgctc agaattgcgt cagtcaagtt cgatctggtc 3601 cttgctattg cacccgttct ccgattacga gtttcattta aatcatgtga gcaaaaggcc 3661 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 3721 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 3781 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 3841 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3901 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3961 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4021 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4081 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4141 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4201 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4261 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4321 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4381 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 4441 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 4501 tctgtctatt tcgttcatcc atagttgcat ttaaatttcc gaactctcca aggccctcgt 4561 cggaaaatct tcaaaccttt cgtccgatcc atcttgcagg ctacctctcg aacgaactat 4621 cgcaagtctc ttggccggcc ttgcgccttg gctattgctt ggcagcgcct atcgccaggt 4681 attactccaa tcccgaatat ccgagatcgg gatcacccga gagaagttca acctacatcc 4741 tcaatcccga tctatccgag atccgaggaa tatcgaaatc ggggcgcgcc tggcctccgc 4801 gccgggtttt ggcgcctccc gcgggcgccc ccctcgtcac ggcgagcgct gccacgtcag 4861 acgaagggcg caggagcgtc ctgatccttc cgcccggacg ctcaggacag cggcccgctg 4921 ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag gacgggactt 4981 gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg aaaagtagtc 5041 ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat gattatataa 5101 ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt cgcggttctt 5161 gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct ggccggggct 5221 ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc caagggctgt 5281 agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg cagcaaaatg 5341 gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa 5401 acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgagccctt cgctaatgcg 5461 ggaaagctct tattcgggtg agatgggctg ggcaccatct ggggaccctg acgtgaagtt 5521 tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt atggcggtgc 5581 cgttgggcag tgcacccgta cctttgggag cgcgcgccct cgtcgtgtcg tgacgtcacc 5641 cgttctgttg gcttataatg cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt 5701 tctccgtcgc aggacgcagg gttcgggcct agggtaggct ctcctgaatc gacaggcgcc 5761 ggacctctgg tgaggggagg gataagtgag gcgtcagttt ctttggtcgg ttttatgtac 5821 ctatcttctt aagtagctga agctccggtt ttgaactatg cgctcggggt tggcgagtgt 5881 gttttgtgaa gttttttagg caccttttga aatgtaatca tttgggtcaa tatgtaattt 5941 tcagtgttag acttgtaaat tgtccgctaa attctggccg tttttggctt ttttgttaga 6001 caacatgggt aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa 6061 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag 6121 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta 6181 caaagatcgt tatgtttatc ggcactttgc atccgccgcg ctcccgattc cggaagtgct 6241 tgacattggg gagttcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt 6301 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc 6361 aatggatgcc atcgctgccg ccgatcttag ccagacgagc gggttcggcc cattcggacc 6421 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca 6481 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 6541 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 6601 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag 6661 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg 6721 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg 6781 atcgccccgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt 6841 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 6901 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgccg ccgtctggac 6961 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag 7021 ggcaaaggaa taagctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga 7081 agatggatgt gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa 7141 cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga 7201 atccagcttg ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc 7261 agcgtaccga tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag 7321 tcctagcttt tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta 7381 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 7441 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg 7501 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 7561 gctttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 7621 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 7681 attcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 7741 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac 7801 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 7861 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 7921 caatggcaac aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc 7981 aacagttgat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 8041 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 8101 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 8161 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 8221 ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc 8281 tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg 8341 cccacttcca tacgtgtcct ccttaccaga aatttatcct taagatcgtt taaactcgac 8401 tctggctcta tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg 8461 ctcgtcgggc atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg 8521 ctcccgacat cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca 8581 gcagcttcag ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg 8641 ggttatgcta tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat 8701 ggatagcgat tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac 8761 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 8821 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 8881 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 8941 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 9001 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 9061 ttggcagtac atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta 9121 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 9181 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 9241 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 9301 agctggttta gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac 9361 ccgccagcgg ccgc //