LOCUS Exported 9425 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA polymerase. ACCESSION . VERSION . KEYWORDS pGBW-m4133616 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9425) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 9425) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9425 /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..2896 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" polyA_signal 2965..3086 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2976..2995) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3030..3049 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 3201..3247 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 3277..3413 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3458..3529 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3755..4343) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3835..3854) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4843..6052 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5889..5908 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 6056..7084 /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 7111..7159 /note="synthetic polyadenylation signal" CDS 7425..8285 /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(7643..7662) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 8298..8434 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 8579..8650 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 8655..8701 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 8719..8767 /note="synthetic polyadenylation signal" enhancer 8848..9151 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 9153..9355 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 9306..9326 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 9424..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 ttcagtcgaa 2821 gaggcgatgg aaaaagaatt ttatcgcggt gagtgcagcc aacagattca aaaagatatc 2881 ctcgtctggt gccctctaag gatccgcagg cctctgctag cttgactgac tgagatacag 2941 cgtaccttca gctcacagac atgataagat acattgatga gtttggacaa accacaacta 3001 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 3061 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 3121 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtattg 3181 gcccatctct atcggtatcg tagcataacc ccttggggcc tctaaacggg tcttgagggg 3241 ttttttgtgc ccctcgggcc ggattgctat ctaccggcat tggcgcagaa aaaaatgcct 3301 gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt 3361 ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct taaggtcgtc 3421 agctatcctg caggcgatct ctcgatttcg atcaagacat tcctttaatg gtcttttctg 3481 gacaccacta ggggtcagaa gtagttcatc aaactttctt ccctccctaa tctcattggt 3541 taccttgggc tatcgaaact taattaacca gtcaagtcag ctacttggcg agatcgactt 3601 gtctgggttt cgactacgct cagaattgcg tcagtcaagt tcgatctggt ccttgctatt 3661 gcacccgttc tccgattacg agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg 3721 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 3781 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 3841 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 3901 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 3961 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 4021 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 4081 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 4141 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 4201 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 4261 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 4321 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 4381 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 4441 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 4501 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 4561 ttcgttcatc catagttgca tttaaatttc cgaactctcc aaggccctcg tcggaaaatc 4621 ttcaaacctt tcgtccgatc catcttgcag gctacctctc gaacgaacta tcgcaagtct 4681 cttggccggc cttgcgcctt ggctattgct tggcagcgcc tatcgccagg tattactcca 4741 atcccgaata tccgagatcg ggatcacccg agagaagttc aacctacatc ctcaatcccg 4801 atctatccga gatccgagga atatcgaaat cggggcgcgc ctggcctccg cgccgggttt 4861 tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc tgccacgtca gacgaagggc 4921 gcaggagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga 4981 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc 5041 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg 5101 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc 5161 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga 5221 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc 5281 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt 5341 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt 5401 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg 5461 ggggcatggt gggcggcaag aacccaaggt cttgagccct tcgctaatgc gggaaagctc 5521 ttattcgggt gagatgggct gggcaccatc tggggaccct gacgtgaagt ttgtcactga 5581 ctggagaact cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg ccgttgggca 5641 gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt 5701 ggcttataat gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg 5761 caggacgcag ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc cggacctctg 5821 gtgaggggag ggataagtga ggcgtcagtt tctttggtcg gttttatgta cctatcttct 5881 taagtagctg aagctccggt tttgaactat gcgctcgggg ttggcgagtg tgttttgtga 5941 agttttttag gcaccttttg aaatgtaatc atttgggtca atatgtaatt ttcagtgtta 6001 gacttgtaaa ttgtccgcta aattctggcc gtttttggct tttttgttag acaacatggg 6061 taaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 6121 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 6181 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 6241 ttatgtttat cggcactttg catccgccgc gctcccgatt ccggaagtgc ttgacattgg 6301 ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 6361 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg caatggatgc 6421 catcgctgcc gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 6481 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 6541 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 6601 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 6661 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 6721 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 6781 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccccg 6841 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 6901 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 6961 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcc gccgtctgga ccgatggctg 7021 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 7081 ataagctagt atgtaagcct agtcttagat aataaaatcg ctatccatcg aagatggatg 7141 tgtgttggtt ttttgtgtgt gtaacgctag gcgcgcctgg tgtaccgaga acgatcctct 7201 cagtgcgagt ctcgacgatc catatcgttg cttggcagtc agccagtcgg aatccagctt 7261 gggacccagg aagtccaatc gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg 7321 atctgtttaa acctagatat tgatagtctg atcggtcaac gtataatcga gtcctagctt 7381 ttgcaaacat ctatcaagag acaggatcag caggaggctt tcgcatgagt attcaacatt 7441 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 7501 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg ggttacatcg 7561 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgctttccaa 7621 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 7681 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag 7741 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 7801 ccatgagtga taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc 7861 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 7921 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 7981 caaccttgcg taaactatta actggcgaac tacttactct agcttcccgg caacagttga 8041 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 8101 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 8161 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 8221 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 8281 ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct 8341 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc 8401 atacgtgtcc tccttaccag aaatttatcc ttaagatcgt ttaaactcga ctctggctct 8461 atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg 8521 catcgaatct cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca 8581 tcttggacca ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca 8641 gctacctctc aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct 8701 atcaatcgtt gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga 8761 ttttattatc taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt 8821 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 8881 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 8941 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 9001 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 9061 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 9121 catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg 9181 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 9241 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 9301 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt 9361 agtgaaccgt cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg 9421 gccgc //