LOCUS Exported 8002 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4133877 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8002) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8002) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8002 /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 1877..1894 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1971..2092 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1982..2001) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2036..2055 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2207..2253 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2283..2419 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2464..2535 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2761..3349) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2841..2860) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3849..5058 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4895..4914 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 5062..5661 /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(5062..5081) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5558..5578 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5688..5736 /note="synthetic polyadenylation signal" CDS 6002..6862 /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(6220..6239) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6875..7011 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7156..7227 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7232..7278 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7296..7344 /note="synthetic polyadenylation signal" enhancer 7425..7728 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7730..7932 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7883..7903 /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 ttcagtcgat gagtgacaaa atcattcatc taaccgatga 1561 ctcctttgat acagacgtgt tgaaggccga tggtgcgata ctggtagatt tctgggctga 1621 atggtgtggc ccgtgcaaga tgatcgcacc cattctggac gagatcgccg acgagtatca 1681 aggaaaatta actgtcgcta agcttaatat agatcagaac ccagggacgg cacctaaata 1741 cggcatcagg ggaataccta ccctcctgct ctttaagaat ggggaagttg ccgcgacaaa 1801 agtgggagcc ctgtctaagg gccagctaaa agagttcttg gacgctaacc tggcaggttc 1861 aggctcgggg cacatgcacc atcaccatca ccatagcagc gggctcgtgc cacgcggcta 1921 agctagcttg actgactgag atacagcgta ccttcagctc acagacatga taagatacat 1981 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 2041 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 2101 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa 2161 gtaaaacctc tacaaatgtg gtattggccc atctctatcg gtatcgtagc ataacccctt 2221 ggggcctcta aacgggtctt gaggggtttt ttgtgcccct cgggccggat tgctatctac 2281 cggcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat 2341 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt 2401 accagaaatt tatccttaag gtcgtcagct atcctgcagg cgatctctcg atttcgatca 2461 agacattcct ttaatggtct tttctggaca ccactagggg tcagaagtag ttcatcaaac 2521 tttcttccct ccctaatctc attggttacc ttgggctatc gaaacttaat taaccagtca 2581 agtcagctac ttggcgagat cgacttgtct gggtttcgac tacgctcaga attgcgtcag 2641 tcaagttcga tctggtcctt gctattgcac ccgttctccg attacgagtt tcatttaaat 2701 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 2761 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 2821 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 2881 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 2941 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 3001 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3061 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3121 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3181 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 3241 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3301 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 3361 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3421 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 3481 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 3541 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta aatttccgaa 3601 ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc ttgcaggcta 3661 cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct attgcttggc 3721 agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat cacccgagag 3781 aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat cgaaatcggg 3841 gcgcgcctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcgtcacggc 3901 gagcgctgcc acgtcagacg aagggcgcag gagcgtcctg atccttccgc ccggacgctc 3961 aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca gcagaaggac 4021 attttaggac gggacttggg tgactctagg gcactggttt tctttccaga gagcggaaca 4081 ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtgaa 4141 cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc gcagccggga 4201 tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg 4261 ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag 4321 agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact gggggttggg 4381 gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg 4441 gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc caaggtcttg 4501 agcccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggc accatctggg 4561 gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg ttgcgggggc 4621 ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt 4681 cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgccggta 4741 ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc 4801 ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt 4861 tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc 4921 tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt 4981 gggtcaatat gtaattttca gtgttagact tgtaaattgt ccgctaaatt ctggccgttt 5041 ttggcttttt tgttagacaa catgaccgag tacaagccca cggtgcgcct cgccacccgc 5101 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 5161 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 5221 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 5281 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 5341 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga gggcctcctg 5401 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 5461 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc cgccgagcgc 5521 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 5581 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 5641 acccgcaagc ccggtgccta ggctagtatg taagcctagt cttagataat aaaatcgcta 5701 tccatcgaag atggatgtgt gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt 5761 accgagaacg atcctctcag tgcgagtctc gacgatccat atcgttgctt ggcagtcagc 5821 cagtcggaat ccagcttggg acccaggaag tccaatcgtc agatattgta ctcaagcctg 5881 gtcacggcag cgtaccgatc tgtttaaacc tagatattga tagtctgatc ggtcaacgta 5941 taatcgagtc ctagcttttg caaacatcta tcaagagaca ggatcagcag gaggctttcg 6001 catgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 6061 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 6121 gcgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 6181 cgaagaacgc tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 6241 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 6301 ggttgagtat tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 6361 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 6421 tggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 6481 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 6541 gcctgtagca atggcaacaa ccttgcgtaa actattaact ggcgaactac ttactctagc 6601 ttcccggcaa cagttgatag actggatgga ggcggataaa gttgcaggac cacttctgcg 6661 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 6721 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 6781 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 6841 ctcactgatt aagcattggt aaccgattct aggtgcattg gcgcagaaaa aaatgcctga 6901 tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc 6961 ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta agatcgttta 7021 aactcgactc tggctctatc gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg 7081 ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt cagcttacct ttttggcagc 7141 gatcgcggct cccgacatct tggaccatta gctccacagg tatcttcttc cctctagtgg 7201 tcataacagc agcttcagct acctctcaat tcaaaaaacc cctcaagacc cgtttagagg 7261 ccccaagggg ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa cacacatcca 7321 tcttcgatgg atagcgattt tattatctaa ctgctgatcg agtgtagcca gatctagtaa 7381 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 7441 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 7501 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 7561 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 7621 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 7681 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt 7741 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 7801 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 7861 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 7921 ataagcagag ctggtttagt gaaccgtcag atcagatctt tgtcgatcct accatccact 7981 cgacacaccc gccagcggcc gc //