LOCUS Exported 9457 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA polymerase. ACCESSION . VERSION . KEYWORDS pGBW-m4133722 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9457) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 9457) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9457 /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 2852..2929 /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" CDS 2957..2977 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 3426..3547 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3437..3456) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3491..3510 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 3662..3708 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 3738..3874 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3919..3990 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(4216..4804) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4296..4315) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 5304..6513 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 6350..6369 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 6517..7116 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(6517..6536) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7013..7033 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 7143..7191 /note="synthetic polyadenylation signal" CDS 7457..8317 /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(7675..7694) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 8330..8466 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 8611..8682 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 8687..8733 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 8751..8799 /note="synthetic polyadenylation signal" enhancer 8880..9183 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 9185..9387 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 9338..9358 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 9456..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 ttcagtcggg 2821 acggcgaatc ccggggctga taaatccctg gaaaaggaga tggaagaaga actttattgc 2881 agtgtctgcg gctaaccggt tcaaaaagat cagttcaagc ggcgccctag attacgacat 2941 tcctacgacc gcctccgaga atctctattt ccagggtgaa tttggcttgg cacaacatga 3001 cgaggccgtc gataacaagt ttaataaaga acagcagaac gctttctatg agatccttca 3061 cttaccaaat ctgaacgaag agcaacgcaa cgctttcatt cagagcctga aagacgatcc 3121 ctcgcagtct gcaaatctcc tggccgaagc caagaagctg aatgacgcac aggctccaaa 3181 agttgataac aagtttaaca aggagcagca gaatgctttc tacgagatat tgcaccttcc 3241 taatctgaat gaggaacaaa gaaatgcatt tatccagtcc ctaaaagacg atccatcaca 3301 gagtgccaac ctgcttgcgg aagccaaaaa gctcaatgat gcccaggccc cgaaagtaga 3361 cgcgaaccat caataagcta gcttgactga ctgagataca gcgtaccttc agctcacaga 3421 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg 3481 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa 3541 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga 3601 ggttttttaa agcaagtaaa acctctacaa atgtggtatt ggcccatctc tatcggtatc 3661 gtagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgtg cccctcgggc 3721 cggattgcta tctaccggca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct 3781 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc 3841 atacgtgtcc tccttaccag aaatttatcc ttaaggtcgt cagctatcct gcaggcgatc 3901 tctcgatttc gatcaagaca ttcctttaat ggtcttttct ggacaccact aggggtcaga 3961 agtagttcat caaactttct tccctcccta atctcattgg ttaccttggg ctatcgaaac 4021 ttaattaacc agtcaagtca gctacttggc gagatcgact tgtctgggtt tcgactacgc 4081 tcagaattgc gtcagtcaag ttcgatctgg tccttgctat tgcacccgtt ctccgattac 4141 gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 4201 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 4261 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 4321 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 4381 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 4441 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 4501 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 4561 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 4621 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 4681 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4741 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 4801 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 4861 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 4921 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 4981 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 5041 atttaaattt ccgaactctc caaggccctc gtcggaaaat cttcaaacct ttcgtccgat 5101 ccatcttgca ggctacctct cgaacgaact atcgcaagtc tcttggccgg ccttgcgcct 5161 tggctattgc ttggcagcgc ctatcgccag gtattactcc aatcccgaat atccgagatc 5221 gggatcaccc gagagaagtt caacctacat cctcaatccc gatctatccg agatccgagg 5281 aatatcgaaa tcggggcgcg cctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc 5341 ccccctcgtc acggcgagcg ctgccacgtc agacgaaggg cgcaggagcg tcctgatcct 5401 tccgcccgga cgctcaggac agcggcccgc tgctcataag actcggcctt agaaccccag 5461 tatcagcaga aggacatttt aggacgggac ttgggtgact ctagggcact ggttttcttt 5521 ccagagagcg gaacaggcga ggaaaagtag tcccttctcg gcgattctgc ggagggatct 5581 ccgtggggcg gtgaacgccg atgattatat aaggacgcgc cgggtgtggc acagctagtt 5641 ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg atcgctgtga tcgtcacttg 5701 gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc cgccgggccg ctcggtggga 5761 cggaagcgtg tggagagacc gccaagggct gtagtctggg tccgcgagca aggttgccct 5821 gaactggggg ttggggggag cgcagcaaaa tggcggctgt tcccgagtct tgaatggaag 5881 acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa 5941 gaacccaagg tcttgagccc ttcgctaatg cgggaaagct cttattcggg tgagatgggc 6001 tgggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc 6061 gtctgttgcg ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg 6121 agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg 6181 ggccacctgc cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc 6241 ctagggtagg ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg 6301 aggcgtcagt ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg 6361 ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt 6421 gaaatgtaat catttgggtc aatatgtaat tttcagtgtt agacttgtaa attgtccgct 6481 aaattctggc cgtttttggc ttttttgtta gacaacatga ccgagtacaa gcccacggtg 6541 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc 6601 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag 6661 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac 6721 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc 6781 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag 6841 atggagggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc 6901 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg 6961 gaggccgccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc 7021 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg 7081 cgcacctggt gcatgacccg caagcccggt gcctaggcta gtatgtaagc ctagtcttag 7141 ataataaaat cgctatccat cgaagatgga tgtgtgttgg ttttttgtgt gtgtaacgct 7201 aggcgcgcct ggtgtaccga gaacgatcct ctcagtgcga gtctcgacga tccatatcgt 7261 tgcttggcag tcagccagtc ggaatccagc ttgggaccca ggaagtccaa tcgtcagata 7321 ttgtactcaa gcctggtcac ggcagcgtac cgatctgttt aaacctagat attgatagtc 7381 tgatcggtca acgtataatc gagtcctagc ttttgcaaac atctatcaag agacaggatc 7441 agcaggaggc tttcgcatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 7501 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 7561 agatcagttg ggtgcgcgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 7621 tgagagtttt cgccccgaag aacgctttcc aatgatgagc acttttaaag ttctgctatg 7681 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 7741 ttctcagaat gacttggttg agtattcacc agtcacagaa aagcatctta cggatggcat 7801 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 7861 acttctgaca acgattggag gaccgaagga gctaaccgct tttttgcaca acatggggga 7921 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 7981 gcgtgacacc acgatgcctg tagcaatggc aacaaccttg cgtaaactat taactggcga 8041 actacttact ctagcttccc ggcaacagtt gatagactgg atggaggcgg ataaagttgc 8101 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 8161 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 8221 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 8281 cgctgagata ggtgcctcac tgattaagca ttggtaaccg attctaggtg cattggcgca 8341 gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc agattcagca 8401 acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc agaaatttat 8461 ccttaagatc gtttaaactc gactctggct ctatcgaatc tccgtcgttt cgagcttacg 8521 cgaacagccg tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct atcgtcagct 8581 tacctttttg gcagcgatcg cggctcccga catcttggac cattagctcc acaggtatct 8641 tcttccctct agtggtcata acagcagctt cagctacctc tcaattcaaa aaacccctca 8701 agacccgttt agaggcccca aggggttatg ctatcaatcg ttgcgttaca cacacaaaaa 8761 accaacacac atccatcttc gatggatagc gattttatta tctaactgct gatcgagtgt 8821 agccagatct agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 8881 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 8941 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 9001 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 9061 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 9121 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 9181 atgctgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 9241 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 9301 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 9361 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcag atctttgtcg 9421 atcctaccat ccactcgaca cacccgccag cggccgc //