LOCUS Exported 7396 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 2'-O-ribose methyltransferase. ACCESSION . VERSION . KEYWORDS pGBW-m4133325 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7396) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7396) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7396 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 902..922 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1271..1288 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1365..1486 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1376..1395) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1430..1449 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1601..1647 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1677..1813 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1858..1929 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2155..2743) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2235..2254) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3243..4452 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4289..4308 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4456..5055 /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(4456..4475) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4952..4972 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5082..5130 /note="synthetic polyadenylation signal" CDS 5396..6256 /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(5614..5633) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6269..6405 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6550..6621 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6626..6672 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6690..6738 /note="synthetic polyadenylation signal" enhancer 6819..7122 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7124..7326 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7277..7297 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgtcc tcacaagcgt ggcagccagg cgtagcaatg cctaatttat acaagatgca 61 gcggatgctc cttgaaaaat gcgacctaca gaactatgga gatagtgcta cattgcccaa 121 ggggataatg atgaatgtgg ccaaatatac ccaactgtgt cagtacctga acactctcac 181 gctggccgtt ccgtacaaca tgagggtgat tcattttggt gccggcagcg acaagggagt 241 cgctcccggg accgctgtgc tgcgacagtg gcttcctaca ggcactctgc tagtggattc 301 tgatttgaat gacttcgtca gcgacgcaga ctcgaccctc atcggagatt gtgcgactgt 361 tcacacagcc aacaaatggg acctgatcat ttccgatatg tatgatccaa agaccaaaaa 421 tgtcacgaag gagaacgact caaaggaggg tttctttacg tacatctgcg ggttcatcca 481 acagaaactt gcactgggcg gaagtgtggc aattaaaata acagaacact cttggaacgc 541 cgacctatac aagcttatgg gtcattttgc ttggtggacc gcctttgtta ctaatgtaaa 601 cgcttcaagc tccgaggcct tcctgatagg ttgtaattat ttaggcaagc ctagagagca 661 gattgatggg tatgtgatgc acgcaaatta catcttctgg cgcaacacaa atccaattca 721 gctcagttct tattcactgt ttgatatgag caaattcccc cttaaactga gaggcactgc 781 cgtcatgtcc ttgaaggaag gacaaatcaa cgatatgatc ttgtctctcc ttagcaaggg 841 gcgtttgatt attagggaga acaatcgggt ggtgatctca tcggacgttc tggtcaacaa 901 tgaaaacctt tactttcagt cgatgagtga caaaatcatt catctaaccg atgactcctt 961 tgatacagac gtgttgaagg ccgatggtgc gatactggta gatttctggg ctgaatggtg 1021 tggcccgtgc aagatgatcg cacccattct ggacgagatc gccgacgagt atcaaggaaa 1081 attaactgtc gctaagctta atatagatca gaacccaggg acggcaccta aatacggcat 1141 caggggaata cctaccctcc tgctctttaa gaatggggaa gttgccgcga caaaagtggg 1201 agccctgtct aagggccagc taaaagagtt cttggacgct aacctggcag gttcaggctc 1261 ggggcacatg caccatcacc atcaccatag cagcgggctc gtgccacgcg gctaagctag 1321 cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat acattgatga 1381 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 1441 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 1501 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa 1561 cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc 1621 tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat 1681 tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga 1741 ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga 1801 aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat 1861 tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt 1921 ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag 1981 ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt 2041 tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg 2101 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2161 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2221 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2281 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2341 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2401 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2461 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2521 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2581 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2641 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2701 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2761 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2821 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 2881 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2941 tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc cgaactctcc 3001 aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag gctacctctc 3061 gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct tggcagcgcc 3121 tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg agagaagttc 3181 aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat cggggcgcgc 3241 ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc 3301 tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca 3361 gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta 3421 ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag 3481 gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga 3541 tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg 3601 tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc 3661 tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg 3721 ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc 3781 gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg 3841 aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgagccct 3901 tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc tggggaccct 3961 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 4021 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 4081 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 4141 cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 4201 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 4261 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 4321 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 4381 atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc gtttttggct 4441 tttttgttag acaacatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 4501 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 4561 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 4621 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 4681 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 4741 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggagggcct cctggcgccg 4801 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag 4861 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggccgccga gcgcgccggg 4921 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 4981 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 5041 aagcccggtg cctaggctag tatgtaagcc tagtcttaga taataaaatc gctatccatc 5101 gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag 5161 aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt cagccagtcg 5221 gaatccagct tgggacccag gaagtccaat cgtcagatat tgtactcaag cctggtcacg 5281 gcagcgtacc gatctgttta aacctagata ttgatagtct gatcggtcaa cgtataatcg 5341 agtcctagct tttgcaaaca tctatcaaga gacaggatca gcaggaggct ttcgcatgag 5401 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 5461 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt 5521 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 5581 acgctttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 5641 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 5701 gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5761 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgattggagg 5821 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5881 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5941 agcaatggca acaaccttgc gtaaactatt aactggcgaa ctacttactc tagcttcccg 6001 gcaacagttg atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 6061 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 6121 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 6181 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 6241 gattaagcat tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac 6301 gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact 6361 tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaagatcg tttaaactcg 6421 actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt 6481 tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc 6541 ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta gtggtcataa 6601 cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta gaggccccaa 6661 ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca tccatcttcg 6721 atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta gtaatcaatt 6781 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 6841 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 6901 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 6961 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 7021 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 7081 acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg gttttggcag 7141 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 7201 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 7261 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 7321 agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc cactcgacac 7381 acccgccagc ggccgc //