LOCUS Exported 7102 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3C-like proteinase. ACCESSION . VERSION . KEYWORDS pGBW-m4133922 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7102) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7102) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7102 /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..1018 /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 1071..1192 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1082..1101) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1136..1155 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1307..1353 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1383..1519 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1564..1635 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1861..2449) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1941..1960) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2949..4158 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3995..4014 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4162..4761 /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(4162..4181) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4658..4678 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4788..4836 /note="synthetic polyadenylation signal" CDS 5102..5962 /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(5320..5339) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5975..6111 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6256..6327 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6332..6378 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6396..6444 /note="synthetic polyadenylation signal" enhancer 6525..6828 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6830..7032 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6983..7003 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7101..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 aagaggcgat ggaaaaagaa 961 ttttatcgcg gtgagtgcag ccaacagatt caaaaagata tcctcgtctg gtgccctcta 1021 agctagcttg actgactgag atacagcgta ccttcagctc acagacatga taagatacat 1081 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 1141 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 1201 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa 1261 gtaaaacctc tacaaatgtg gtattggccc atctctatcg gtatcgtagc ataacccctt 1321 ggggcctcta aacgggtctt gaggggtttt ttgtgcccct cgggccggat tgctatctac 1381 cggcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat 1441 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt 1501 accagaaatt tatccttaag gtcgtcagct atcctgcagg cgatctctcg atttcgatca 1561 agacattcct ttaatggtct tttctggaca ccactagggg tcagaagtag ttcatcaaac 1621 tttcttccct ccctaatctc attggttacc ttgggctatc gaaacttaat taaccagtca 1681 agtcagctac ttggcgagat cgacttgtct gggtttcgac tacgctcaga attgcgtcag 1741 tcaagttcga tctggtcctt gctattgcac ccgttctccg attacgagtt tcatttaaat 1801 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 1861 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 1921 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 1981 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 2041 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 2101 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 2161 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 2221 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 2281 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 2341 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 2401 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 2461 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 2521 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 2581 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 2641 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta aatttccgaa 2701 ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc ttgcaggcta 2761 cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct attgcttggc 2821 agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat cacccgagag 2881 aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat cgaaatcggg 2941 gcgcgcctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcgtcacggc 3001 gagcgctgcc acgtcagacg aagggcgcag gagcgtcctg atccttccgc ccggacgctc 3061 aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca gcagaaggac 3121 attttaggac gggacttggg tgactctagg gcactggttt tctttccaga gagcggaaca 3181 ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtgaa 3241 cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc gcagccggga 3301 tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg 3361 ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag 3421 agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact gggggttggg 3481 gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg 3541 gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc caaggtcttg 3601 agcccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggc accatctggg 3661 gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg ttgcgggggc 3721 ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt 3781 cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgccggta 3841 ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc 3901 ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt 3961 tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc 4021 tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt 4081 gggtcaatat gtaattttca gtgttagact tgtaaattgt ccgctaaatt ctggccgttt 4141 ttggcttttt tgttagacaa catgaccgag tacaagccca cggtgcgcct cgccacccgc 4201 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 4261 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 4321 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 4381 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 4441 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga gggcctcctg 4501 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 4561 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc cgccgagcgc 4621 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 4681 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 4741 acccgcaagc ccggtgccta ggctagtatg taagcctagt cttagataat aaaatcgcta 4801 tccatcgaag atggatgtgt gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt 4861 accgagaacg atcctctcag tgcgagtctc gacgatccat atcgttgctt ggcagtcagc 4921 cagtcggaat ccagcttggg acccaggaag tccaatcgtc agatattgta ctcaagcctg 4981 gtcacggcag cgtaccgatc tgtttaaacc tagatattga tagtctgatc ggtcaacgta 5041 taatcgagtc ctagcttttg caaacatcta tcaagagaca ggatcagcag gaggctttcg 5101 catgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 5161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 5221 gcgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 5281 cgaagaacgc tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 5341 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 5401 ggttgagtat tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 5461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 5521 tggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 5581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 5641 gcctgtagca atggcaacaa ccttgcgtaa actattaact ggcgaactac ttactctagc 5701 ttcccggcaa cagttgatag actggatgga ggcggataaa gttgcaggac cacttctgcg 5761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 5821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 5881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 5941 ctcactgatt aagcattggt aaccgattct aggtgcattg gcgcagaaaa aaatgcctga 6001 tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc 6061 ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta agatcgttta 6121 aactcgactc tggctctatc gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg 6181 ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt cagcttacct ttttggcagc 6241 gatcgcggct cccgacatct tggaccatta gctccacagg tatcttcttc cctctagtgg 6301 tcataacagc agcttcagct acctctcaat tcaaaaaacc cctcaagacc cgtttagagg 6361 ccccaagggg ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa cacacatcca 6421 tcttcgatgg atagcgattt tattatctaa ctgctgatcg agtgtagcca gatctagtaa 6481 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 6541 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 6601 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 6661 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 6721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 6781 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt 6841 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 6901 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 6961 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 7021 ataagcagag ctggtttagt gaaccgtcag atcagatctt tgtcgatcct accatccact 7081 cgacacaccc gccagcggcc gc //