LOCUS Exported 7487 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3C-like proteinase. ACCESSION . VERSION . KEYWORDS pGBW-m4133821 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7487) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7487) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7487 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 920..940 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 941..958 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1027..1148 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1038..1057) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1092..1111 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1263..1309 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1339..1475 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1520..1591 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1817..2405) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1897..1916) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2905..4114 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3951..3970 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4118..5146 /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 5173..5221 /note="synthetic polyadenylation signal" CDS 5487..6347 /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(5705..5724) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6360..6496 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6641..6712 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6717..6763 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6781..6829 /note="synthetic polyadenylation signal" enhancer 6910..7213 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7215..7417 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7368..7388 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7486..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatgggt tttagaaaaa tggcattccc atctggtaaa gttgagggtt gtatggtaca 61 agtaacttgt ggtacaacta cacttaacgg tctttggctt gatgacgtag tttactgtcc 121 aagacatgtg atctgcacct ctgaagacat gcttaaccct aattatgaag atttactcat 181 tcgtaagtct aatcataatt tcttggtaca ggctggtaat gttcaactca gggttattgg 241 acattctatg caaaattgtg tacttaagct taaggttgat acagccaatc ctaagacacc 301 taagtataag tttgttcgca ttcaaccagg acagactttt tcagtgttag cttgttacaa 361 tggttcacca tctggtgttt accaatgtgc tatgaggccc aatttcacta ttaagggttc 421 attccttaat ggttcatgtg gtagtgttgg ttttaacata gattatgact gtgtctcttt 481 ttgttacatg caccatatgg aattaccaac tggagttcat gctggcacag acttagaagg 541 taacttttat ggaccttttg ttgacaggca aacagcacaa gcagctggta cggacacaac 601 tattacagtt aatgttttag cttggttgta cgctgctgtt ataaatggag acaggtggtt 661 tctcaatcga tttaccacaa ctcttaatga ctttaacctt gtggctatga agtacaatta 721 tgaacctcta acacaagacc atgttgacat actaggacct ctttctgctc aaactggaat 781 tgccgtttta gatatgtgtg cttcattaaa agaattactg caaaatggta tgaatggacg 841 taccatattg ggtagtgctt tattagaaga tgaatttaca ccttttgatg ttgttagaca 901 atgctcaggt gttactttcg aaaaccttta ctttcagtcg caccatcacc atcaccatta 961 aggatccgca ggcctctgct agcttgactg actgagatac agcgtacctt cagctcacag 1021 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 1081 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 1141 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg 1201 aggtttttta aagcaagtaa aacctctaca aatgtggtat tggcccatct ctatcggtat 1261 cgtagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgt gcccctcggg 1321 ccggattgct atctaccggc attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc 1381 ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact tgcccacttc 1441 catacgtgtc ctccttacca gaaatttatc cttaaggtcg tcagctatcc tgcaggcgat 1501 ctctcgattt cgatcaagac attcctttaa tggtcttttc tggacaccac taggggtcag 1561 aagtagttca tcaaactttc ttccctccct aatctcattg gttaccttgg gctatcgaaa 1621 cttaattaac cagtcaagtc agctacttgg cgagatcgac ttgtctgggt ttcgactacg 1681 ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta ttgcacccgt tctccgatta 1741 cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 1801 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 1861 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 1921 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 1981 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 2041 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 2101 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 2161 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 2221 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 2281 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 2341 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 2401 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 2461 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 2521 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 2581 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 2641 catttaaatt tccgaactct ccaaggccct cgtcggaaaa tcttcaaacc tttcgtccga 2701 tccatcttgc aggctacctc tcgaacgaac tatcgcaagt ctcttggccg gccttgcgcc 2761 ttggctattg cttggcagcg cctatcgcca ggtattactc caatcccgaa tatccgagat 2821 cgggatcacc cgagagaagt tcaacctaca tcctcaatcc cgatctatcc gagatccgag 2881 gaatatcgaa atcggggcgc gcctggcctc cgcgccgggt tttggcgcct cccgcgggcg 2941 cccccctcgt cacggcgagc gctgccacgt cagacgaagg gcgcaggagc gtcctgatcc 3001 ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca 3061 gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt 3121 tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc 3181 tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt 3241 tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt 3301 ggtgagtagc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg 3361 acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc 3421 tgaactgggg gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa 3481 gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca 3541 agaacccaag gtcttgagcc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg 3601 ctgggcacca tctggggacc ctgacgtgaa gtttgtcact gactggagaa ctcggtttgt 3661 cgtctgttgc gggggcggca gttatggcgg tgccgttggg cagtgcaccc gtacctttgg 3721 gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg ttggcttata atgcagggtg 3781 gggccacctg ccggtaggtg tgcggtaggc ttttctccgt cgcaggacgc agggttcggg 3841 cctagggtag gctctcctga atcgacaggc gccggacctc tggtgagggg agggataagt 3901 gaggcgtcag tttctttggt cggttttatg tacctatctt cttaagtagc tgaagctccg 3961 gttttgaact atgcgctcgg ggttggcgag tgtgttttgt gaagtttttt aggcaccttt 4021 tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt tagacttgta aattgtccgc 4081 taaattctgg ccgtttttgg cttttttgtt agacaacatg ggtaaaaagc ctgaactcac 4141 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 4201 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt 4261 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt 4321 tgcatccgcc gcgctcccga ttccggaagt gcttgacatt ggggagttca gcgagagcct 4381 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga 4441 actgcccgct gttctgcagc cggtcgcgga ggcaatggat gccatcgctg ccgccgatct 4501 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg 4561 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga 4621 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga 4681 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga 4741 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata 4801 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg 4861 ctacttcgag cggaggcatc cggagcttgc aggatcgccc cggctccggg cgtatatgct 4921 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc 4981 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac 5041 acaaatcgcc cgcagaagcg ccgccgtctg gaccgatggc tgtgtagaag tactcgccga 5101 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataagcta gtatgtaagc 5161 ctagtcttag ataataaaat cgctatccat cgaagatgga tgtgtgttgg ttttttgtgt 5221 gtgtaacgct aggcgcgcct ggtgtaccga gaacgatcct ctcagtgcga gtctcgacga 5281 tccatatcgt tgcttggcag tcagccagtc ggaatccagc ttgggaccca ggaagtccaa 5341 tcgtcagata ttgtactcaa gcctggtcac ggcagcgtac cgatctgttt aaacctagat 5401 attgatagtc tgatcggtca acgtataatc gagtcctagc ttttgcaaac atctatcaag 5461 agacaggatc agcaggaggc tttcgcatga gtattcaaca tttccgtgtc gcccttattc 5521 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 5581 aagatgctga agatcagttg ggtgcgcgag tgggttacat cgaactggat ctcaacagcg 5641 gtaagatcct tgagagtttt cgccccgaag aacgctttcc aatgatgagc acttttaaag 5701 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 5761 gcatacacta ttctcagaat gacttggttg agtattcacc agtcacagaa aagcatctta 5821 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 5881 cggccaactt acttctgaca acgattggag gaccgaagga gctaaccgct tttttgcaca 5941 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 6001 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaaccttg cgtaaactat 6061 taactggcga actacttact ctagcttccc ggcaacagtt gatagactgg atggaggcgg 6121 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 6181 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 6241 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 6301 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaaccg attctaggtg 6361 cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc 6421 agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc 6481 agaaatttat ccttaagatc gtttaaactc gactctggct ctatcgaatc tccgtcgttt 6541 cgagcttacg cgaacagccg tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct 6601 atcgtcagct tacctttttg gcagcgatcg cggctcccga catcttggac cattagctcc 6661 acaggtatct tcttccctct agtggtcata acagcagctt cagctacctc tcaattcaaa 6721 aaacccctca agacccgttt agaggcccca aggggttatg ctatcaatcg ttgcgttaca 6781 cacacaaaaa accaacacac atccatcttc gatggatagc gattttatta tctaactgct 6841 gatcgagtgt agccagatct agtaatcaat tacggggtca ttagttcata gcccatatat 6901 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 6961 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 7021 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 7081 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 7141 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 7201 cgctattacc atgctgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 7261 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 7321 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 7381 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcag 7441 atctttgtcg atcctaccat ccactcgaca cacccgccag cggccgc //