LOCUS Exported 7511 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3C-like proteinase. ACCESSION . VERSION . KEYWORDS pGBW-m4133414 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7511) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7511) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7511 /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..982 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" polyA_signal 1051..1172 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1062..1081) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1116..1135 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1287..1333 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1363..1499 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1544..1615 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1841..2429) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1921..1940) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2929..4138 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3975..3994 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4142..5170 /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 5197..5245 /note="synthetic polyadenylation signal" CDS 5511..6371 /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(5729..5748) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6384..6520 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6665..6736 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6741..6787 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6805..6853 /note="synthetic polyadenylation signal" enhancer 6934..7237 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7239..7441 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7392..7412 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7510..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 gggaaaccta tacccaatcc 961 acttctcggc ttagattcta cctaaggatc cgcaggcctc tgctagcttg actgactgag 1021 atacagcgta ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca 1081 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 1141 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 1201 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg 1261 gtattggccc atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt 1321 gaggggtttt ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa 1381 atgcctgatg cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata 1441 cggcttcccc aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag 1501 gtcgtcagct atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct 1561 tttctggaca ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc 1621 attggttacc ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat 1681 cgacttgtct gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt 1741 gctattgcac ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc 1801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 1861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 1921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 1981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 2041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 2101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 2161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 2221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 2281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 2341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 2401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 2461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 2521 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 2581 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 2641 gtctatttcg ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg 2701 aaaatcttca aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc 2761 aagtctcttg gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt 2821 actccaatcc cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca 2881 atcccgatct atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc 2941 gggttttggc gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg 3001 aagggcgcag gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc 3061 ataagactcg gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg 3121 tgactctagg gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct 3181 tctcggcgat tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga 3241 cgcgccgggt gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt 3301 tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc 3361 gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt 3421 ctgggtccgc gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg 3481 gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca 3541 aggtgggggg catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga 3601 aagctcttat tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt 3661 cactgactgg agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt 3721 tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt 3781 tctgttggct tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct 3841 ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga 3901 cctctggtga ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta 3961 tcttcttaag tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt 4021 ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca 4081 gtgttagact tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa 4141 catgggtaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt 4201 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt 4261 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa 4321 agatcgttat gtttatcggc actttgcatc cgccgcgctc ccgattccgg aagtgcttga 4381 cattggggag ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac 4441 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggcaat 4501 ggatgccatc gctgccgccg atcttagcca gacgagcggg ttcggcccat tcggaccgca 4561 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt 4621 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga 4681 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt 4741 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga 4801 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt 4861 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc 4921 gccccggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt 4981 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc 5041 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgccgccg tctggaccga 5101 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc 5161 aaaggaataa gctagtatgt aagcctagtc ttagataata aaatcgctat ccatcgaaga 5221 tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga 5281 tcctctcagt gcgagtctcg acgatccata tcgttgcttg gcagtcagcc agtcggaatc 5341 cagcttggga cccaggaagt ccaatcgtca gatattgtac tcaagcctgg tcacggcagc 5401 gtaccgatct gtttaaacct agatattgat agtctgatcg gtcaacgtat aatcgagtcc 5461 tagcttttgc aaacatctat caagagacag gatcagcagg aggctttcgc atgagtattc 5521 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 5581 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt 5641 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgct 5701 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 5761 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtatt 5821 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 5881 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatt ggaggaccga 5941 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 6001 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 6061 tggcaacaac cttgcgtaaa ctattaactg gcgaactact tactctagct tcccggcaac 6121 agttgataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 6181 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 6241 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 6301 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 6361 agcattggta accgattcta ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc 6421 gcgtcttata ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc 6481 acttccatac gtgtcctcct taccagaaat ttatccttaa gatcgtttaa actcgactct 6541 ggctctatcg aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc 6601 gtcgggcatc gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc 6661 ccgacatctt ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca 6721 gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt 6781 tatgctatca atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga 6841 tagcgatttt attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg 6901 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 6961 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 7021 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 7081 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 7141 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 7201 gcagtacatc tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacat 7261 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 7321 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 7381 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 7441 tggtttagtg aaccgtcaga tcagatcttt gtcgatccta ccatccactc gacacacccg 7501 ccagcggccg c //