LOCUS Exported 8423 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp2. ACCESSION . VERSION . KEYWORDS pGBW-m4134436 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8423) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8423) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8423 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 306..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 577..586 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2500..2520 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2869..2886 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 2963..3084 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2974..2993) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3028..3047 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 3199..3245 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 3275..3411 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3456..3527 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3753..4341) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3833..3852) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4841..6050 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5887..5906 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 6054..6653 /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(6054..6073) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6550..6570 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6680..6728 /note="synthetic polyadenylation signal" CDS 6994..7854 /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(7212..7231) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 7867..8003 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 8155..8226 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 8231..8277 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 8295..8343 /note="synthetic polyadenylation signal" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catgctgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc 541 gatcctacca tccactcgac acacccgcca gcggccgcca ccatggccta cacaagatat 601 gtggataaca atttttgtgg gccggacgga taccctttag aatgcatcaa agatttgctt 661 gcaagggcgg gcaaggcttc gtgcactctg tccgagcagc tggacttcat tgataccaag 721 cgcggtgttt attgttgccg agagcatgaa cacgagatag cctggtatac ggaacggtca 781 gagaaaagtt acgaactcca aaccccattc gagatcaagc tagctaaaaa gtttgacact 841 ttcaacggcg aatgtcccaa ttttgtcttt cccctgaact ctattatcaa aacaatacag 901 cctcgggtgg agaagaagaa attagacgga ttcatggggc gtattaggag cgtataccca 961 gtggccagcc cgaatgaatg caaccagatg tgtctttcca cactgatgaa gtgcgatcac 1021 tgtggcgaga cttcttggca aaccggtgac ttcgtaaaag cgacatgcga gttttgtggt 1081 accgagaatc tgactaagga gggagctacc acatgcggct atttgccaca gaatgcagtg 1141 gtcaagatct actgtcctgc atgccataac tctgaagtcg gccccgagca ctcgctcgcc 1201 gaatatcaca atgagagcgg gctaaaaact atactgagga aaggaggtcg caccatcgcc 1261 tttgggggct gcgttttctc ctacgtgggg tgtcataaca aatgtgccta ttgggttccc 1321 agagcctccg ctaacattgg gtgcaatcat acgggagtgg tcggggaagg ctcagaaggc 1381 ttgaacgata acctccttga aattctgcaa aaggagaagg tgaatatcaa tatcgttggc 1441 gacttcaagt taaatgagga aatcgcgatt attctggcaa gtttttctgc ctcaactagc 1501 gcctttgtag agacagtgaa aggactcgat tataaagctt tcaaacagat agtcgagagt 1561 tgtggtaact tcaaggttac aaagggcaag gctaaaaagg gtgcttggaa cattggagag 1621 cagaagtcaa tcttgagtcc actgtacgcg tttgcatctg aggccgcccg tgtggtcagg 1681 agcatattca gtcgaaccct agaaaccgca cagaatagcg tgcgggtatt acagaaggcg 1741 gctattacta ttctcgacgg tatctcgcag tattctctga gactgatcga cgccatgatg 1801 tttacaagcg atctggcaac gaataactta gtggtcatgg cgtacataac cggaggggtc 1861 gtgcaactta cctcccagtg gctaacgaac atctttggga cggtttacga gaagctaaaa 1921 cctgtgctgg actggctcga agagaaattc aaagaaggcg tggaattttt gcgtgacggg 1981 tgggagatcg taaagttcat atcaacatgt gcttgcgaaa tcgttggcgg acaaatcgta 2041 acttgcgcaa aagagattaa agaatcggtg cagaccttct ttaaacttgt taacaaattc 2101 ctggctttgt gtgccgattc tattatcatc gggggagcaa agcttaaagc cctaaattta 2161 ggcgagacat ttgtgacgca cagtaagggc ctgtatagaa agtgcgtgaa gagtcgcgaa 2221 gaaaccggac tgctcatgcc cttaaaagcg ccaaaagaaa taattttcct ggaaggggag 2281 acattgccca ctgaagtgct cactgaggaa gtagttctga aaactgggga tctgcaacct 2341 ttagagcagc cgacctccga ggccgtcgag gcacctctcg tgggaacccc ggtctgtatt 2401 aatgggttga tgctgctcga gattaaggat accgaaaaat actgcgcact agcccccaac 2461 atgatggtga ctaacaacac atttaccctc aagggagggg aaaaccttta ctttcagtcg 2521 atgagtgaca aaatcattca tctaaccgat gactcctttg atacagacgt gttgaaggcc 2581 gatggtgcga tactggtaga tttctgggct gaatggtgtg gcccgtgcaa gatgatcgca 2641 cccattctgg acgagatcgc cgacgagtat caaggaaaat taactgtcgc taagcttaat 2701 atagatcaga acccagggac ggcacctaaa tacggcatca ggggaatacc taccctcctg 2761 ctctttaaga atggggaagt tgccgcgaca aaagtgggag ccctgtctaa gggccagcta 2821 aaagagttct tggacgctaa cctggcaggt tcaggctcgg ggcacatgca ccatcaccat 2881 caccatagca gcgggctcgt gccacgcggc taagctagct tgactgactg agatacagcg 2941 taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac cacaactaga 3001 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 3061 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 3121 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtattggc 3181 ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc ttgaggggtt 3241 ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa aaatgcctga 3301 tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc 3361 ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag 3421 ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt cttttctgga 3481 caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc tcattggtta 3541 ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag atcgacttgt 3601 ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc ttgctattgc 3661 acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc 3721 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3781 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3841 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3901 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 3961 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 4021 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 4081 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 4141 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 4201 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 4261 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 4321 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 4381 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 4441 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 4501 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 4561 cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc ggaaaatctt 4621 caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc gcaagtctct 4681 tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta ttactccaat 4741 cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct caatcccgat 4801 ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg ccgggttttg 4861 gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga cgaagggcgc 4921 aggagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc tcataagact 4981 cggccttaga accccagtat cagcagaagg acattttagg acgggacttg ggtgactcta 5041 gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc cttctcggcg 5101 attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag gacgcgccgg 5161 gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg tttgtggatc 5221 gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt tcgtggccgc 5281 cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc 5341 gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg cggctgttcc 5401 cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg 5461 ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg gaaagctctt 5521 attcgggtga gatgggctgg gcaccatctg gggaccctga cgtgaagttt gtcactgact 5581 ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc gttgggcagt 5641 gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc gttctgttgg 5701 cttataatgc agggtggggc cacctgccgg taggtgtgcg gtaggctttt ctccgtcgca 5761 ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg acaggcgccg gacctctggt 5821 gaggggaggg ataagtgagg cgtcagtttc tttggtcggt tttatgtacc tatcttctta 5881 agtagctgaa gctccggttt tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag 5941 ttttttaggc accttttgaa atgtaatcat ttgggtcaat atgtaatttt cagtgttaga 6001 cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt tttgttagac aacatgaccg 6061 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 6121 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 6181 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 6241 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 6301 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 6361 tggccgcgca gcaacagatg gagggcctcc tggcgccgca ccggcccaag gagcccgcgt 6421 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg 6481 tcgtgctccc cggagtggag gccgccgagc gcgccggggt gcccgccttc ctggagacct 6541 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 6601 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc taggctagta 6661 tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt gtgttggttt 6721 tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc 6781 tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg ggacccagga 6841 agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga tctgtttaaa 6901 cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt tgcaaacatc 6961 tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc 7021 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 7081 aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga actggatctc 7141 aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat gatgagcact 7201 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 7261 ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt cacagaaaag 7321 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 7381 aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct aaccgctttt 7441 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 7501 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aaccttgcgt 7561 aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat agactggatg 7621 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 7681 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 7741 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 7801 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaaccgatt 7861 ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac 7921 atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct 7981 ccttaccaga aatttatcct taagatcccg aatcgtttaa actcgactct ggctctatcg 8041 aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc 8101 gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc ccgacatctt 8161 ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca gcttcagcta 8221 cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctatca 8281 atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga tagcgatttt 8341 attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt 8401 catagcccat atatggagtt ccg //