LOCUS Exported 5790 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4133483 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5790) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5790) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5790 /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..1606 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" polyA_signal 1659..1780 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1670..1689) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1724..1743 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1895..1941 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1971..2107 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2152..2223 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2449..3037) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2529..2548) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 3783..4643 /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(4001..4020) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 4656..4792 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 4944..5015 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5020..5066 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5084..5132 /note="synthetic polyadenylation signal" enhancer 5213..5516 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5518..5720 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 5671..5691 /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 ttcagtcgaa gaggcgatgg aaaaagaatt ttatcgcggt 1561 gagtgcagcc aacagattca aaaagatatc ctcgtctggt gccctctaag ctagcttgac 1621 tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg atgagtttgg 1681 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 1741 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 1801 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 1861 caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg ggcctctaaa 1921 cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg gcattggcgc 1981 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc 2041 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta 2101 tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag acattccttt 2161 aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt tcttccctcc 2221 ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag tcagctactt 2281 ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc 2341 tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca tgtgagcaaa 2401 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 2461 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 2521 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 2581 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 2641 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 2701 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 2761 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 2821 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 2881 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 2941 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 3001 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 3061 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 3121 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 3181 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 3241 agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact ctccaaggcc 3301 ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc tctcgaacga 3361 actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag cgcctatcgc 3421 caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa gttcaaccta 3481 catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc gcgcctggtg 3541 taccgagaac gatcctctca gtgcgagtct cgacgatcca tatcgttgct tggcagtcag 3601 ccagtcggaa tccagcttgg gacccaggaa gtccaatcgt cagatattgt actcaagcct 3661 ggtcacggca gcgtaccgat ctgtttaaac ctagatattg atagtctgat cggtcaacgt 3721 ataatcgagt cctagctttt gcaaacatct atcaagagac aggatcagca ggaggctttc 3781 gcatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3841 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3901 cgcgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3961 ccgaagaacg ctttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4021 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4081 tggttgagta ttcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4141 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 4201 ttggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4261 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4321 tgcctgtagc aatggcaaca accttgcgta aactattaac tggcgaacta cttactctag 4381 cttcccggca acagttgata gactggatgg aggcggataa agttgcagga ccacttctgc 4441 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4501 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4561 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4621 cctcactgat taagcattgg taaccgattc taggtgcatt ggcgcagaaa aaaatgcctg 4681 atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc 4741 cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt aagatcccga 4801 atcgtttaaa ctcgactctg gctctatcga atctccgtcg tttcgagctt acgcgaacag 4861 ccgtggcgct catttgctcg tcgggcatcg aatctcgtca gctatcgtca gcttaccttt 4921 ttggcagcga tcgcggctcc cgacatcttg gaccattagc tccacaggta tcttcttccc 4981 tctagtggtc ataacagcag cttcagctac ctctcaattc aaaaaacccc tcaagacccg 5041 tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca 5101 cacatccatc ttcgatggat agcgatttta ttatctaact gctgatcgag tgtagccaga 5161 tctagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 5221 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 5281 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 5341 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 5401 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 5461 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatgctga 5521 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 5581 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 5641 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 5701 aggtctatat aagcagagct ggtttagtga accgtcagat cagatctttg tcgatcctac 5761 catccactcg acacacccgc cagcggccgc //