LOCUS Exported 7661 bp ds-DNA circular SYN 17-MAR-2022 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4134223 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7661) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7661) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 17, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7661 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1508..1528 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1529..1570 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" polyA_signal 1623..1744 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1634..1653) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1688..1707 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1859..1905 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1935..2071 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2116..2187 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2413..3001) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2493..2512) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3501..4710 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4547..4566 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4714..5313 /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(4714..4733) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5210..5230 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5340..5388 /note="synthetic polyadenylation signal" CDS 5654..6514 /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(5872..5891) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6527..6663 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6815..6886 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6891..6937 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6955..7003 /note="synthetic polyadenylation signal" enhancer 7084..7387 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7389..7591 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7542..7562 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgaag attgtaaata actggttgaa acaactaata aaggttactt tagtgttcct 61 gtttgtcgcc gcaatcttct atcttatcac cccggtgcac gtgatgagta aacatacaga 121 cttttcttca gaaattatcg ggtacaaggc tatcgatggc ggtgtaacgc gcgacattgc 181 ctccacagat acctgcttcg cgaacaaaca cgctgacttt gatacttggt tcagccagcg 241 aggaggctcg tacaccaatg acaaagcctg tccactgatc gcagccgtga taactagaga 301 ggttggattc gtcgtccctg ggctccccgg gacaattctc cgtacaacca acggcgactt 361 tctgcatttt ctgccccggg tgttctccgc agtcggtaat atctgttata ctccatctaa 421 gctcattgag tacacggatt ttgctaccag cgcgtgcgtg ttggccgcag aatgcaccat 481 atttaaggac gcttctggaa aacctgtgcc ctattgttac gataccaatg tgcttgaggg 541 ctcagttgcc tatgaaagtc tgaggccaga cactagatac gttttaatgg atggaagcat 601 catccagttc ccaaacacgt atctggaggg gtccgtgcgg gtcgtgacca cattcgattc 661 agaatactgc cgccacggta catgcgagag gtctgaggct ggcgtatgtg tgtcaactag 721 cggtcgatgg gttctaaata acgattatta tcgttctctg cccggcgttt tttgtggagt 781 ggacgccgtt aaccttttga ccaacatgtt tacacctctc attcagccca ttggtgcact 841 ggatatctcc gcgagtattg tggctggggg aatagtcgcc attgtcgtaa cctgcttggc 901 atactatttc atgcgtttca ggcgagcttt tggcgaatac tcacatgtcg tggccttcaa 961 tactcttctg tttctgatga gcttcacggt actctgtctg accccagtat acagtttcct 1021 acctggggtg tattctgtga tctacttata cctcacattt tacctgacca atgacgtgtc 1081 ctttctggcc cacatccaat ggatggttat gttcacgccg ctcgtgcctt tctggattac 1141 aatcgcttat ataatctgta ttagcacgaa gcatttctat tggttctttt ccaactactt 1201 aaagcggcgc gtcgtattta atggggtatc gtttagcact tttgaggaag ccgcgctctg 1261 cactttcttg cttaataagg agatgtattt gaaattaaga tctgacgttc tgcttccttt 1321 aacacagtac aacagatacc tggcgttgta taacaaatac aaatatttct cgggagccat 1381 ggatacaact tcctatagag aggctgcttg ttgccacctt gcaaaggcac tcaacgactt 1441 cagtaacagc ggcagtgatg tgctttatca gccccctcag acttcaataa catccgccgt 1501 cctccaagaa aacctttact ttcagtcggg gaaacctata cccaatccac ttctcggctt 1561 agattctacc taagctagct tgactgactg agatacagcg taccttcagc tcacagacat 1621 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 1681 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 1741 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt 1801 tttttaaagc aagtaaaacc tctacaaatg tggtattggc ccatctctat cggtatcgta 1861 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc ctcgggccgg 1921 attgctatct accggcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat 1981 actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata 2041 cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag ctatcctgca ggcgatctct 2101 cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg ggtcagaagt 2161 agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta tcgaaactta 2221 attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg actacgctca 2281 gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc cgattacgag 2341 tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2401 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2461 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 2521 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2581 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 2641 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2701 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2761 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2821 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 2881 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2941 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3001 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 3061 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3121 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3181 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcatt 3241 taaatttccg aactctccaa ggccctcgtc ggaaaatctt caaacctttc gtccgatcca 3301 tcttgcaggc tacctctcga acgaactatc gcaagtctct tggccggcct tgcgccttgg 3361 ctattgcttg gcagcgccta tcgccaggta ttactccaat cccgaatatc cgagatcggg 3421 atcacccgag agaagttcaa cctacatcct caatcccgat ctatccgaga tccgaggaat 3481 atcgaaatcg gggcgcgcct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc 3541 cctcgtcacg gcgagcgctg ccacgtcaga cgaagggcgc aggagcgtcc tgatccttcc 3601 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat 3661 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca 3721 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg 3781 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg 3841 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg 3901 agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg 3961 aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa 4021 ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg 4081 cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa 4141 cccaaggtct tgagcccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg 4201 gcaccatctg gggaccctga cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc 4261 tgttgcgggg gcggcagtta tggcggtgcc gttgggcagt gcacccgtac ctttgggagc 4321 gcgcgccctc gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc agggtggggc 4381 cacctgccgg taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta 4441 gggtaggctc tcctgaatcg acaggcgccg gacctctggt gaggggaggg ataagtgagg 4501 cgtcagtttc tttggtcggt tttatgtacc tatcttctta agtagctgaa gctccggttt 4561 tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa 4621 atgtaatcat ttgggtcaat atgtaatttt cagtgttaga cttgtaaatt gtccgctaaa 4681 ttctggccgt ttttggcttt tttgttagac aacatgaccg agtacaagcc cacggtgcgc 4741 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 4801 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 4861 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 4921 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 4981 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 5041 gagggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 5101 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 5161 gccgccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 5221 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 5281 acctggtgca tgacccgcaa gcccggtgcc taggctagta tgtaagccta gtcttagata 5341 ataaaatcgc tatccatcga agatggatgt gtgttggttt tttgtgtgtg taacgctagg 5401 cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc 5461 ttggcagtca gccagtcgga atccagcttg ggacccagga agtccaatcg tcagatattg 5521 tactcaagcc tggtcacggc agcgtaccga tctgtttaaa cctagatatt gatagtctga 5581 tcggtcaacg tataatcgag tcctagcttt tgcaaacatc tatcaagaga caggatcagc 5641 aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 5701 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 5761 tcagttgggt gcgcgagtgg gttacatcga actggatctc aacagcggta agatccttga 5821 gagttttcgc cccgaagaac gctttccaat gatgagcact tttaaagttc tgctatgtgg 5881 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 5941 tcagaatgac ttggttgagt attcaccagt cacagaaaag catcttacgg atggcatgac 6001 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 6061 tctgacaacg attggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 6121 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 6181 tgacaccacg atgcctgtag caatggcaac aaccttgcgt aaactattaa ctggcgaact 6241 acttactcta gcttcccggc aacagttgat agactggatg gaggcggata aagttgcagg 6301 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 6361 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 6421 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 6481 tgagataggt gcctcactga ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa 6541 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg 6601 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct 6661 taagatcccg aatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct 6721 tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc 6781 agcttacctt tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt 6841 atcttcttcc ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc 6901 ctcaagaccc gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca 6961 aaaaaccaac acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga 7021 gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat atatggagtt 7081 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 7141 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 7201 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 7261 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 7321 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 7381 taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg 7441 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca 7501 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg 7561 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt 7621 gtcgatccta ccatccactc gacacacccg ccagcggccg c //