LOCUS Exported 7996 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4133665 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7996) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7996) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7996 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1502..1522 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1871..1888 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1965..2086 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1976..1995) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2030..2049 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2201..2247 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2277..2413 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2458..2529 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2755..3343) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2835..2854) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3843..5052 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4889..4908 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 5056..5655 /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(5056..5075) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5552..5572 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5682..5730 /note="synthetic polyadenylation signal" CDS 5996..6856 /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(6214..6233) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6869..7005 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7150..7221 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7226..7272 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7290..7338 /note="synthetic polyadenylation signal" enhancer 7419..7722 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7724..7926 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7877..7897 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgatt gttaataatt ggttgaagca gttaattaaa gttacacttg tgttcctttt 61 tgttgctgct attttctatt taataacacc tgttcatgtc atgtctaaac atactgactt 121 ttcaagtgaa atcataggat acaaggctat tgatggtggt gtcactcgtg acatagcatc 181 tacagatact tgttttgcta acaaacatgc tgattttgac acatggttta gccagcgtgg 241 tggtagttat actaatgaca aagcttgccc attgattgct gcagtcataa caagagaagt 301 gggttttgtc gtgcctggtt tgcctggcac gatattacgc acaactaatg gtgacttttt 361 gcatttctta cctagagttt ttagtgcagt tggtaacatc tgttacacac catcaaaact 421 tatagagtac actgactttg caacatcagc ttgtgttttg gctgctgaat gtacaatttt 481 taaagatgct tctggtaagc cagtaccata ttgttatgat accaatgtac tagaaggttc 541 tgttgcttat gaaagtttac gccctgacac acgttatgtg ctcatggatg gctctattat 601 tcaatttcct aacacctacc ttgaaggttc tgttagagtg gtaacaactt ttgattctga 661 gtactgtagg cacggcactt gtgaaagatc agaagctggt gtttgtgtat ctactagtgg 721 tagatgggta cttaacaatg attattacag atctttacca ggagttttct gtggtgtaga 781 tgctgtaaat ttacttacta atatgtttac accactaatt caacctattg gtgctttgga 841 catatcagca tctatagtag ctggtggtat tgtagctatc gtagtaacat gccttgccta 901 ctattttatg aggtttagaa gagcttttgg tgaatacagt catgtagttg cctttaatac 961 tttactattc cttatgtcat tcactgtact ctgtttaaca ccagtttact cattcttacc 1021 tggtgtttat tctgttattt acttgtactt gacattttat cttactaatg atgtttcttt 1081 tttagcacat attcagtgga tggttatgtt cacaccttta gtacctttct ggataacaat 1141 tgcttatatc atttgtattt ccacaaagca tttctattgg ttctttagta attacctaaa 1201 gagacgtgta gtctttaatg gtgtttcctt tagtactttt gaagaagctg cgctgtgcac 1261 ctttttgtta aataaagaaa tgtatctaaa gttgcgtagt gatgtgctat tacctcttac 1321 gcaatataat agatacttag ctctttataa taagtacaag tattttagtg gagcaatgga 1381 tacaactagc tacagagaag ctgcttgttg tcatctcgca aaggctctca atgacttcag 1441 taactcaggt tctgatgttc tttaccaacc accacaaacc tctatcacct cagctgtttt 1501 ggaaaacctt tactttcagt cgatgagtga caaaatcatt catctaaccg atgactcctt 1561 tgatacagac gtgttgaagg ccgatggtgc gatactggta gatttctggg ctgaatggtg 1621 tggcccgtgc aagatgatcg cacccattct ggacgagatc gccgacgagt atcaaggaaa 1681 attaactgtc gctaagctta atatagatca gaacccaggg acggcaccta aatacggcat 1741 caggggaata cctaccctcc tgctctttaa gaatggggaa gttgccgcga caaaagtggg 1801 agccctgtct aagggccagc taaaagagtt cttggacgct aacctggcag gttcaggctc 1861 ggggcacatg caccatcacc atcaccatag cagcgggctc gtgccacgcg gctaagctag 1921 cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat acattgatga 1981 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 2041 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 2101 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa 2161 cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc 2221 tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat 2281 tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga 2341 ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga 2401 aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat 2461 tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt 2521 ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag 2581 ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt 2641 tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg 2701 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2761 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2821 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2881 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2941 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3001 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 3061 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 3121 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 3181 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 3241 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 3301 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 3361 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 3421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 3481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 3541 tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc cgaactctcc 3601 aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag gctacctctc 3661 gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct tggcagcgcc 3721 tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg agagaagttc 3781 aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat cggggcgcgc 3841 ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc 3901 tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca 3961 gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta 4021 ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag 4081 gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga 4141 tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg 4201 tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc 4261 tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg 4321 ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc 4381 gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg 4441 aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgagccct 4501 tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc tggggaccct 4561 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 4621 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 4681 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 4741 cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 4801 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 4861 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 4921 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 4981 atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc gtttttggct 5041 tttttgttag acaacatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 5101 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 5161 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 5221 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 5281 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 5341 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggagggcct cctggcgccg 5401 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag 5461 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggccgccga gcgcgccggg 5521 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 5581 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 5641 aagcccggtg cctaggctag tatgtaagcc tagtcttaga taataaaatc gctatccatc 5701 gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag 5761 aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt cagccagtcg 5821 gaatccagct tgggacccag gaagtccaat cgtcagatat tgtactcaag cctggtcacg 5881 gcagcgtacc gatctgttta aacctagata ttgatagtct gatcggtcaa cgtataatcg 5941 agtcctagct tttgcaaaca tctatcaaga gacaggatca gcaggaggct ttcgcatgag 6001 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 6061 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt 6121 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 6181 acgctttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 6241 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 6301 gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 6361 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgattggagg 6421 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 6481 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 6541 agcaatggca acaaccttgc gtaaactatt aactggcgaa ctacttactc tagcttcccg 6601 gcaacagttg atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 6661 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 6721 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 6781 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 6841 gattaagcat tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac 6901 gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact 6961 tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaagatcg tttaaactcg 7021 actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt 7081 tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc 7141 ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta gtggtcataa 7201 cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta gaggccccaa 7261 ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca tccatcttcg 7321 atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta gtaatcaatt 7381 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 7441 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 7501 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 7561 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 7621 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 7681 acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg gttttggcag 7741 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 7801 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 7861 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 7921 agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc cactcgacac 7981 acccgccagc ggccgc //