LOCUS Exported 7579 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3C-like proteinase. ACCESSION . VERSION . KEYWORDS pGBW-m4133870 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7579) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7579) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7579 /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 974..1051 /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" CDS 1079..1099 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 1548..1669 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1559..1578) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1613..1632 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1784..1830 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1860..1996 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2041..2112 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2338..2926) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2418..2437) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3426..4635 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4472..4491 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4639..5238 /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(4639..4658) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5135..5155 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5265..5313 /note="synthetic polyadenylation signal" CDS 5579..6439 /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(5797..5816) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6452..6588 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6733..6804 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6809..6855 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6873..6921 /note="synthetic polyadenylation signal" enhancer 7002..7305 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7307..7509 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7460..7480 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7578..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 ggacggcgaa tcccggggct 961 gataaatccc tggaaaagga gatggaagaa gaactttatt gcagtgtctg cggctaaccg 1021 gttcaaaaag atcagttcaa gcggcgccct agattacgac attcctacga ccgcctccga 1081 gaatctctat ttccagggtg aatttggctt ggcacaacat gacgaggccg tcgataacaa 1141 gtttaataaa gaacagcaga acgctttcta tgagatcctt cacttaccaa atctgaacga 1201 agagcaacgc aacgctttca ttcagagcct gaaagacgat ccctcgcagt ctgcaaatct 1261 cctggccgaa gccaagaagc tgaatgacgc acaggctcca aaagttgata acaagtttaa 1321 caaggagcag cagaatgctt tctacgagat attgcacctt cctaatctga atgaggaaca 1381 aagaaatgca tttatccagt ccctaaaaga cgatccatca cagagtgcca acctgcttgc 1441 ggaagccaaa aagctcaatg atgcccaggc cccgaaagta gacgcgaacc atcaataagc 1501 tagcttgact gactgagata cagcgtacct tcagctcaca gacatgataa gatacattga 1561 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg 1621 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 1681 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta 1741 aaacctctac aaatgtggta ttggcccatc tctatcggta tcgtagcata accccttggg 1801 gcctctaaac gggtcttgag gggttttttg tgcccctcgg gccggattgc tatctaccgg 1861 cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc 1921 agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc 1981 agaaatttat ccttaaggtc gtcagctatc ctgcaggcga tctctcgatt tcgatcaaga 2041 cattccttta atggtctttt ctggacacca ctaggggtca gaagtagttc atcaaacttt 2101 cttccctccc taatctcatt ggttaccttg ggctatcgaa acttaattaa ccagtcaagt 2161 cagctacttg gcgagatcga cttgtctggg tttcgactac gctcagaatt gcgtcagtca 2221 agttcgatct ggtccttgct attgcacccg ttctccgatt acgagtttca tttaaatcat 2281 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 2341 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 2401 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 2461 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 2521 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 2581 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 2641 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 2701 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 2761 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 2821 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 2881 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 2941 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 3001 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 3061 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 3121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcatttaaat ttccgaactc 3181 tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg atccatcttg caggctacct 3241 ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc cttggctatt gcttggcagc 3301 gcctatcgcc aggtattact ccaatcccga atatccgaga tcgggatcac ccgagagaag 3361 ttcaacctac atcctcaatc ccgatctatc cgagatccga ggaatatcga aatcggggcg 3421 cgcctggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcg tcacggcgag 3481 cgctgccacg tcagacgaag ggcgcaggag cgtcctgatc cttccgcccg gacgctcagg 3541 acagcggccc gctgctcata agactcggcc ttagaacccc agtatcagca gaaggacatt 3601 ttaggacggg acttgggtga ctctagggca ctggttttct ttccagagag cggaacaggc 3661 gaggaaaagt agtcccttct cggcgattct gcggagggat ctccgtgggg cggtgaacgc 3721 cgatgattat ataaggacgc gccgggtgtg gcacagctag ttccgtcgca gccgggattt 3781 gggtcgcggt tcttgtttgt ggatcgctgt gatcgtcact tggtgagtag cgggctgctg 3841 ggctggccgg ggctttcgtg gccgccgggc cgctcggtgg gacggaagcg tgtggagaga 3901 ccgccaaggg ctgtagtctg ggtccgcgag caaggttgcc ctgaactggg ggttgggggg 3961 agcgcagcaa aatggcggct gttcccgagt cttgaatgga agacgcttgt gaggcgggct 4021 gtgaggtcgt tgaaacaagg tggggggcat ggtgggcggc aagaacccaa ggtcttgagc 4081 ccttcgctaa tgcgggaaag ctcttattcg ggtgagatgg gctgggcacc atctggggac 4141 cctgacgtga agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc 4201 agttatggcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt 4261 gtcgtgacgt cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt 4321 gtgcggtagg cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg 4381 aatcgacagg cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg 4441 tcggttttat gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg 4501 gggttggcga gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg 4561 tcaatatgta attttcagtg ttagacttgt aaattgtccg ctaaattctg gccgtttttg 4621 gcttttttgt tagacaacat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 4681 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 4741 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 4801 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 4861 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 4921 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaggg cctcctggcg 4981 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 5041 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggccgc cgagcgcgcc 5101 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 5161 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 5221 cgcaagcccg gtgcctaggc tagtatgtaa gcctagtctt agataataaa atcgctatcc 5281 atcgaagatg gatgtgtgtt ggttttttgt gtgtgtaacg ctaggcgcgc ctggtgtacc 5341 gagaacgatc ctctcagtgc gagtctcgac gatccatatc gttgcttggc agtcagccag 5401 tcggaatcca gcttgggacc caggaagtcc aatcgtcaga tattgtactc aagcctggtc 5461 acggcagcgt accgatctgt ttaaacctag atattgatag tctgatcggt caacgtataa 5521 tcgagtccta gcttttgcaa acatctatca agagacagga tcagcaggag gctttcgcat 5581 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 5641 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcgcg 5701 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 5761 agaacgcttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 5821 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 5881 tgagtattca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 5941 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgattgg 6001 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 6061 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 6121 tgtagcaatg gcaacaacct tgcgtaaact attaactggc gaactactta ctctagcttc 6181 ccggcaacag ttgatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 6241 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 6301 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 6361 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 6421 actgattaag cattggtaac cgattctagg tgcattggcg cagaaaaaaa tgcctgatgc 6481 gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca 6541 acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaaga tcgtttaaac 6601 tcgactctgg ctctatcgaa tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc 6661 atttgctcgt cgggcatcga atctcgtcag ctatcgtcag cttacctttt tggcagcgat 6721 cgcggctccc gacatcttgg accattagct ccacaggtat cttcttccct ctagtggtca 6781 taacagcagc ttcagctacc tctcaattca aaaaacccct caagacccgt ttagaggccc 6841 caaggggtta tgctatcaat cgttgcgtta cacacacaaa aaaccaacac acatccatct 6901 tcgatggata gcgattttat tatctaactg ctgatcgagt gtagccagat ctagtaatca 6961 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 7021 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 7081 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 7141 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 7201 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 7261 cctacttggc agtacatcta cgtattagtc atcgctatta ccatgctgat gcggttttgg 7321 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 7381 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 7441 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 7501 agcagagctg gtttagtgaa ccgtcagatc agatctttgt cgatcctacc atccactcga 7561 cacacccgcc agcggccgc //