LOCUS Exported 7078 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 2'-O-ribose methyltransferase. ACCESSION . VERSION . KEYWORDS pGBW-m4133678 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7078) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7078) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7078 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 896..916 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 917..994 /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 1047..1168 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1058..1077) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1112..1131 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1283..1329 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1359..1495 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1540..1611 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1837..2425) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1917..1936) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2925..4134 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3971..3990 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4138..4737 /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(4138..4157) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4634..4654 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4764..4812 /note="synthetic polyadenylation signal" CDS 5078..5938 /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(5296..5315) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5951..6087 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6232..6303 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6308..6354 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6372..6420 /note="synthetic polyadenylation signal" enhancer 6501..6804 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6806..7008 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6959..6979 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgagt caagcgtggc aaccgggtgt tgctatgcct aatctttaca aaatgcaaag 61 aatgctatta gaaaagtgtg accttcaaaa ttatggtgat agtgcaacat tacctaaagg 121 cataatgatg aatgtcgcaa aatatactca actgtgtcaa tatttaaaca cattaacatt 181 agctgtaccc tataatatga gagttataca ttttggtgct ggttctgata aaggagttgc 241 accaggtaca gctgttttaa gacagtggtt gcctacgggt acgctgcttg tcgattcaga 301 tcttaatgac tttgtctctg atgcagattc aactttgatt ggtgattgtg caactgtaca 361 tacagctaat aaatgggatc tcattattag tgatatgtac gaccctaaga ctaaaaatgt 421 tacaaaagaa aatgactcta aagagggttt tttcacttac atttgtgggt ttatacaaca 481 aaagctagct cttggaggtt ccgtggctat aaagataaca gaacattctt ggaatgctga 541 tctttataag ctcatgggac acttcgcatg gtggacagcc tttgttacta atgtgaatgc 601 gtcatcatct gaagcatttt taattggatg taattatctt ggcaaaccac gcgaacaaat 661 agatggttat gtcatgcatg caaattacat attttggagg aatacaaatc caattcagtt 721 gtcttcctat tctttatttg acatgagtaa atttcccctt aaattaaggg gtactgctgt 781 tatgtcttta aaagaaggtc aaatcaatga tatgatttta tctcttctta gtaaaggtag 841 acttataatt agagaaaaca acagagttgt tatttctagt gatgttcttg ttaacgaaaa 901 cctttacttt cagtcgaaga ggcgatggaa aaagaatttt atcgcggtga gtgcagccaa 961 cagattcaaa aagatatcct cgtctggtgc cctctaagct agcttgactg actgagatac 1021 agcgtacctt cagctcacag acatgataag atacattgat gagtttggac aaaccacaac 1081 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt 1141 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca 1201 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat 1261 tggcccatct ctatcggtat cgtagcataa ccccttgggg cctctaaacg ggtcttgagg 1321 ggttttttgt gcccctcggg ccggattgct atctaccggc attggcgcag aaaaaaatgc 1381 ctgatgcgac gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc 1441 ttccccaact tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaaggtcg 1501 tcagctatcc tgcaggcgat ctctcgattt cgatcaagac attcctttaa tggtcttttc 1561 tggacaccac taggggtcag aagtagttca tcaaactttc ttccctccct aatctcattg 1621 gttaccttgg gctatcgaaa cttaattaac cagtcaagtc agctacttgg cgagatcgac 1681 ttgtctgggt ttcgactacg ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta 1741 ttgcacccgt tctccgatta cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa 1801 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 1861 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 1921 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 1981 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 2041 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 2101 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 2161 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 2221 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 2281 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 2341 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 2401 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 2461 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 2521 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 2581 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 2641 atttcgttca tccatagttg catttaaatt tccgaactct ccaaggccct cgtcggaaaa 2701 tcttcaaacc tttcgtccga tccatcttgc aggctacctc tcgaacgaac tatcgcaagt 2761 ctcttggccg gccttgcgcc ttggctattg cttggcagcg cctatcgcca ggtattactc 2821 caatcccgaa tatccgagat cgggatcacc cgagagaagt tcaacctaca tcctcaatcc 2881 cgatctatcc gagatccgag gaatatcgaa atcggggcgc gcctggcctc cgcgccgggt 2941 tttggcgcct cccgcgggcg cccccctcgt cacggcgagc gctgccacgt cagacgaagg 3001 gcgcaggagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg ctgctcataa 3061 gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga cttgggtgac 3121 tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta gtcccttctc 3181 ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata taaggacgcg 3241 ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg 3301 gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg gctttcgtgg 3361 ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc tgtagtctgg 3421 gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa atggcggctg 3481 ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt 3541 ggggggcatg gtgggcggca agaacccaag gtcttgagcc cttcgctaat gcgggaaagc 3601 tcttattcgg gtgagatggg ctgggcacca tctggggacc ctgacgtgaa gtttgtcact 3661 gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg tgccgttggg 3721 cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg 3781 ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt 3841 cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc 3901 tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt 3961 cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt 4021 gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt 4081 tagacttgta aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacaacatg 4141 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc 4201 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc 4261 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc 4321 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc 4381 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc 4441 cggctggccg cgcagcaaca gatggagggc ctcctggcgc cgcaccggcc caaggagccc 4501 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc 4561 gccgtcgtgc tccccggagt ggaggccgcc gagcgcgccg gggtgcccgc cttcctggag 4621 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac 4681 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctaggct 4741 agtatgtaag cctagtctta gataataaaa tcgctatcca tcgaagatgg atgtgtgttg 4801 gttttttgtg tgtgtaacgc taggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg 4861 agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag cttgggaccc 4921 aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta ccgatctgtt 4981 taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag cttttgcaaa 5041 catctatcaa gagacaggat cagcaggagg ctttcgcatg agtattcaac atttccgtgt 5101 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 5161 ggtgaaagta aaagatgctg aagatcagtt gggtgcgcga gtgggttaca tcgaactgga 5221 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc caatgatgag 5281 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 5341 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga 5401 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 5461 tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc 5521 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 5581 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacctt 5641 gcgtaaacta ttaactggcg aactacttac tctagcttcc cggcaacagt tgatagactg 5701 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 5761 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 5821 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 5881 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaacc 5941 gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc 6001 ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg 6061 tcctccttac cagaaattta tccttaagat cgtttaaact cgactctggc tctatcgaat 6121 ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc gggcatcgaa 6181 tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg acatcttgga 6241 ccattagctc cacaggtatc ttcttccctc tagtggtcat aacagcagct tcagctacct 6301 ctcaattcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctatcaatc 6361 gttgcgttac acacacaaaa aaccaacaca catccatctt cgatggatag cgattttatt 6421 atctaactgc tgatcgagtg tagccagatc tagtaatcaa ttacggggtc attagttcat 6481 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 6541 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 6601 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 6661 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 6721 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 6781 gtattagtca tcgctattac catgctgatg cggttttggc agtacatcaa tgggcgtgga 6841 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 6901 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 6961 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac 7021 cgtcagatca gatctttgtc gatcctacca tccactcgac acacccgcca gcggccgc //