LOCUS Exported 7392 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA polymerase. ACCESSION . VERSION . KEYWORDS pGBW-m4133763 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7392) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7392) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7392 /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 3167..3184 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 3261..3382 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3272..3291) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3326..3345 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 3497..3543 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 3573..3709 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3754..3825 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(4051..4639) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4131..4150) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 5385..6245 /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(5603..5622) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6258..6394 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6546..6617 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6622..6668 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6686..6734 /note="synthetic polyadenylation signal" enhancer 6815..7118 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7120..7322 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7273..7293 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7391..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 ttcagtcgat 2821 gagtgacaaa atcattcatc taaccgatga ctcctttgat acagacgtgt tgaaggccga 2881 tggtgcgata ctggtagatt tctgggctga atggtgtggc ccgtgcaaga tgatcgcacc 2941 cattctggac gagatcgccg acgagtatca aggaaaatta actgtcgcta agcttaatat 3001 agatcagaac ccagggacgg cacctaaata cggcatcagg ggaataccta ccctcctgct 3061 ctttaagaat ggggaagttg ccgcgacaaa agtgggagcc ctgtctaagg gccagctaaa 3121 agagttcttg gacgctaacc tggcaggttc aggctcgggg cacatgcacc atcaccatca 3181 ccatagcagc gggctcgtgc cacgcggcta agctagcttg actgactgag atacagcgta 3241 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat 3301 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 3361 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 3421 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc 3481 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 3541 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg 3601 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc 3661 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct 3721 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca 3781 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc 3841 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct 3901 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac 3961 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag 4021 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 4081 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 4141 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 4201 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 4261 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 4321 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 4381 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 4441 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 4501 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 4561 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4621 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4681 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4741 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4801 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4861 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca 4921 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg 4981 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc 5041 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct 5101 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg tgtaccgaga acgatcctct 5161 cagtgcgagt ctcgacgatc catatcgttg cttggcagtc agccagtcgg aatccagctt 5221 gggacccagg aagtccaatc gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg 5281 atctgtttaa acctagatat tgatagtctg atcggtcaac gtataatcga gtcctagctt 5341 ttgcaaacat ctatcaagag acaggatcag caggaggctt tcgcatgagt attcaacatt 5401 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 5461 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg ggttacatcg 5521 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgctttccaa 5581 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 5641 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag 5701 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 5761 ccatgagtga taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc 5821 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 5881 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 5941 caaccttgcg taaactatta actggcgaac tacttactct agcttcccgg caacagttga 6001 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 6061 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 6121 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 6181 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 6241 ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct 6301 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc 6361 atacgtgtcc tccttaccag aaatttatcc ttaagatccc gaatcgttta aactcgactc 6421 tggctctatc gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct 6481 cgtcgggcat cgaatctcgt cagctatcgt cagcttacct ttttggcagc gatcgcggct 6541 cccgacatct tggaccatta gctccacagg tatcttcttc cctctagtgg tcataacagc 6601 agcttcagct acctctcaat tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg 6661 ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa cacacatcca tcttcgatgg 6721 atagcgattt tattatctaa ctgctgatcg agtgtagcca gatctagtaa tcaattacgg 6781 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 6841 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 6901 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 6961 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 7021 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 7081 ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt tggcagtaca 7141 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 7201 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 7261 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 7321 ctggtttagt gaaccgtcag atcagatctt tgtcgatcct accatccact cgacacaccc 7381 gccagcggcc gc //