LOCUS Exported 5781 bp ds-DNA circular SYN 13-JAN-2022 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4134196 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5781) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5781) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 13, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5781 /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" CDS 2086..2106 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2110..2175 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 2228..2349 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2239..2258) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2293..2312 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2464..2510 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2540..2676 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2721..2792 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3018..3606) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3098..3117) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 4352..5212 /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(4570..4589) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5225..5361 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5513..5584 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5589..5635 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5653..5701 /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 ccatgaagat tgtaaataac 601 tggttgaaac aactaataaa ggttacttta gtgttcctgt ttgtcgccgc aatcttctat 661 cttatcaccc cggtgcacgt gatgagtaaa catacagact tttcttcaga aattatcggg 721 tacaaggcta tcgatggcgg tgtaacgcgc gacattgcct ccacagatac ctgcttcgcg 781 aacaaacacg ctgactttga tacttggttc agccagcgag gaggctcgta caccaatgac 841 aaagcctgtc cactgatcgc agccgtgata actagagagg ttggattcgt cgtccctggg 901 ctccccggga caattctccg tacaaccaac ggcgactttc tgcattttct gccccgggtg 961 ttctccgcag tcggtaatat ctgttatact ccatctaagc tcattgagta cacggatttt 1021 gctaccagcg cgtgcgtgtt ggccgcagaa tgcaccatat ttaaggacgc ttctggaaaa 1081 cctgtgccct attgttacga taccaatgtg cttgagggct cagttgccta tgaaagtctg 1141 aggccagaca ctagatacgt tttaatggat ggaagcatca tccagttccc aaacacgtat 1201 ctggaggggt ccgtgcgggt cgtgaccaca ttcgattcag aatactgccg ccacggtaca 1261 tgcgagaggt ctgaggctgg cgtatgtgtg tcaactagcg gtcgatgggt tctaaataac 1321 gattattatc gttctctgcc cggcgttttt tgtggagtgg acgccgttaa ccttttgacc 1381 aacatgttta cacctctcat tcagcccatt ggtgcactgg atatctccgc gagtattgtg 1441 gctgggggaa tagtcgccat tgtcgtaacc tgcttggcat actatttcat gcgtttcagg 1501 cgagcttttg gcgaatactc acatgtcgtg gccttcaata ctcttctgtt tctgatgagc 1561 ttcacggtac tctgtctgac cccagtatac agtttcctac ctggggtgta ttctgtgatc 1621 tacttatacc tcacatttta cctgaccaat gacgtgtcct ttctggccca catccaatgg 1681 atggttatgt tcacgccgct cgtgcctttc tggattacaa tcgcttatat aatctgtatt 1741 agcacgaagc atttctattg gttcttttcc aactacttaa agcggcgcgt cgtatttaat 1801 ggggtatcgt ttagcacttt tgaggaagcc gcgctctgca ctttcttgct taataaggag 1861 atgtatttga aattaagatc tgacgttctg cttcctttaa cacagtacaa cagatacctg 1921 gcgttgtata acaaatacaa atatttctcg ggagccatgg atacaacttc ctatagagag 1981 gctgcttgtt gccaccttgc aaaggcactc aacgacttca gtaacagcgg cagtgatgtg 2041 ctttatcagc cccctcagac ttcaataaca tccgccgtcc tccaagaaaa cctttacttt 2101 cagtcgatgg attataagga ccacgatgga gactacaaag atcatgacat tgattacaag 2161 gacgacgatg acaaataagc tagcttgact gactgagata cagcgtacct tcagctcaca 2221 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 2281 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 2341 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 2401 gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc tctatcggta 2461 tcgtagcata accccttggg gcctctaaac gggtcttgag gggttttttg tgcccctcgg 2521 gccggattgc tatctaccgg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt 2581 cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt 2641 ccatacgtgt cctccttacc agaaatttat ccttaaggtc gtcagctatc ctgcaggcga 2701 tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca ctaggggtca 2761 gaagtagttc atcaaacttt cttccctccc taatctcatt ggttaccttg ggctatcgaa 2821 acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg tttcgactac 2881 gctcagaatt gcgtcagtca agttcgatct ggtccttgct attgcacccg ttctccgatt 2941 acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3001 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3061 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 3121 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 3181 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 3241 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 3301 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 3361 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 3421 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 3481 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 3541 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 3601 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 3661 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 3721 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 3781 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 3841 gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg 3901 atccatcttg caggctacct ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc 3961 cttggctatt gcttggcagc gcctatcgcc aggtattact ccaatcccga atatccgaga 4021 tcgggatcac ccgagagaag ttcaacctac atcctcaatc ccgatctatc cgagatccga 4081 ggaatatcga aatcggggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc 4141 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag 4201 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc 4261 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta 4321 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct 4381 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 4441 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa 4501 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt 4561 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 4621 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca 4681 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 4741 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt 4801 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 4861 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa 4921 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga 4981 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 5041 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 5101 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 5161 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct 5221 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat 5281 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc 5341 ttaccagaaa tttatcctta agatcccgaa tcgtttaaac tcgactctgg ctctatcgaa 5401 tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc atttgctcgt cgggcatcga 5461 atctcgtcag ctatcgtcag cttacctttt tggcagcgat cgcggctccc gacatcttgg 5521 accattagct ccacaggtat cttcttccct ctagtggtca taacagcagc ttcagctacc 5581 tctcaattca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctatcaat 5641 cgttgcgtta cacacacaaa aaaccaacac acatccatct tcgatggata gcgattttat 5701 tatctaactg ctgatcgagt gtagccagat ctagtaatca attacggggt cattagttca 5761 tagcccatat atggagttcc g //