LOCUS Exported 7841 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 2'-O-ribose methyltransferase. ACCESSION . VERSION . KEYWORDS pGBW-m4133335 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7841) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7841) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7841 /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 1381..1502 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1392..1411) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1446..1465 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1617..1663 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1693..1829 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1874..1945 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2171..2759) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2251..2270) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3259..4468 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4305..4324 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4472..5500 /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 5527..5575 /note="synthetic polyadenylation signal" CDS 5841..6701 /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(6059..6078) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6714..6850 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6995..7066 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7071..7117 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7135..7183 /note="synthetic polyadenylation signal" enhancer 7264..7567 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7569..7771 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7722..7742 /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 gctaaggatc 1321 cgcaggcctc tgctagcttg actgactgag atacagcgta ccttcagctc acagacatga 1381 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 1441 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 1501 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt 1561 tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc atctctatcg gtatcgtagc 1621 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgtgcccct cgggccggat 1681 tgctatctac cggcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac 1741 tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg 1801 tgtcctcctt accagaaatt tatccttaag gtcgtcagct atcctgcagg cgatctctcg 1861 atttcgatca agacattcct ttaatggtct tttctggaca ccactagggg tcagaagtag 1921 ttcatcaaac tttcttccct ccctaatctc attggttacc ttgggctatc gaaacttaat 1981 taaccagtca agtcagctac ttggcgagat cgacttgtct gggtttcgac tacgctcaga 2041 attgcgtcag tcaagttcga tctggtcctt gctattgcac ccgttctccg attacgagtt 2101 tcatttaaat catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 2161 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 2221 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 2281 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 2341 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 2401 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 2461 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 2521 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 2581 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 2641 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 2701 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 2761 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 2821 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 2881 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 2941 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta 3001 aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc 3061 ttgcaggcta cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct 3121 attgcttggc agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat 3181 cacccgagag aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat 3241 cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc 3301 tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag gagcgtcctg atccttccgc 3361 ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca 3421 gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt tctttccaga 3481 gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg 3541 gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc 3601 gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag 3661 tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa 3721 gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact 3781 gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct 3841 tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc 3901 caaggtcttg agcccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggc 3961 accatctggg gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg 4021 ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc 4081 gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca 4141 cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg 4201 gtaggctctc ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg 4261 tcagtttctt tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg 4321 aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat 4381 gtaatcattt gggtcaatat gtaattttca gtgttagact tgtaaattgt ccgctaaatt 4441 ctggccgttt ttggcttttt tgttagacaa catgggtaaa aagcctgaac tcaccgcgac 4501 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 4561 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 4621 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 4681 cgccgcgctc ccgattccgg aagtgcttga cattggggag ttcagcgaga gcctgaccta 4741 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 4801 cgctgttctg cagccggtcg cggaggcaat ggatgccatc gctgccgccg atcttagcca 4861 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 4921 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 4981 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 5041 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 5101 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 5161 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 5221 cgagcggagg catccggagc ttgcaggatc gccccggctc cgggcgtata tgctccgcat 5281 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 5341 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 5401 cgcccgcaga agcgccgccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 5461 aaaccgacgc cccagcactc gtccgagggc aaaggaataa gctagtatgt aagcctagtc 5521 ttagataata aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa 5581 cgctaggcgc gcctggtgta ccgagaacga tcctctcagt gcgagtctcg acgatccata 5641 tcgttgcttg gcagtcagcc agtcggaatc cagcttggga cccaggaagt ccaatcgtca 5701 gatattgtac tcaagcctgg tcacggcagc gtaccgatct gtttaaacct agatattgat 5761 agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc aaacatctat caagagacag 5821 gatcagcagg aggctttcgc atgagtattc aacatttccg tgtcgccctt attccctttt 5881 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 5941 ctgaagatca gttgggtgcg cgagtgggtt acatcgaact ggatctcaac agcggtaaga 6001 tccttgagag ttttcgcccc gaagaacgct ttccaatgat gagcactttt aaagttctgc 6061 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 6121 actattctca gaatgacttg gttgagtatt caccagtcac agaaaagcat cttacggatg 6181 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 6241 acttacttct gacaacgatt ggaggaccga aggagctaac cgcttttttg cacaacatgg 6301 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 6361 acgagcgtga caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg 6421 gcgaactact tactctagct tcccggcaac agttgataga ctggatggag gcggataaag 6481 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 6541 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 6601 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 6661 agatcgctga gataggtgcc tcactgatta agcattggta accgattcta ggtgcattgg 6721 cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc 6781 agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat 6841 ttatccttaa gatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct 6901 tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc 6961 agcttacctt tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt 7021 atcttcttcc ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc 7081 ctcaagaccc gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca 7141 aaaaaccaac acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga 7201 gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat atatggagtt 7261 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 7321 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 7381 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 7441 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 7501 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 7561 taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg 7621 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca 7681 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg 7741 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt 7801 gtcgatccta ccatccactc gacacacccg ccagcggccg c //