LOCUS Exported 7505 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp6. ACCESSION . VERSION . KEYWORDS pGBW-m4133615 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7505) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7505) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7505 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 878..898 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 899..976 /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 1045..1166 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1056..1075) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1110..1129 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1281..1327 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1357..1493 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1538..1609 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1835..2423) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1915..1934) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2923..4132 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3969..3988 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4136..5164 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" polyA_signal 5191..5239 /note="synthetic polyadenylation signal" CDS 5505..6365 /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(5723..5742) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6378..6514 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6659..6730 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6735..6781 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6799..6847 /note="synthetic polyadenylation signal" enhancer 6928..7231 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7233..7435 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7386..7406 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgagc gccgttaagc ggacaatcaa agggacccat cactggttgc tccttactat 61 actgacgtct ttactggtct tggtacagag tacacaatgg tcgcttttct tttttctgta 121 cgagaatgca ttcctcccat ttgcgatggg aatcattgct atgtccgcct tcgctatgat 181 gttcgtgaag cacaaacatg ccttcctttg cctgtttctc ctaccctcac tggcaaccgt 241 ggcctatttt aacatggtgt acatgcctgc gagctgggtc atgcgaatta tgacttggct 301 ggacatggtt gatacctccc tatctggctt caagttaaag gactgtgtga tgtatgcatc 361 agctgtagtg ctgctaatcc tgatgacagc tcgcacagtc tacgatgacg gtgccaggag 421 agtgtggact ctgatgaatg tcctcacctt agtttataaa gtatactatg gcaacgctct 481 cgatcaggcc attagtatgt gggcattgat aatctccgtg acgagtaact acagcggagt 541 ggtaaccact gtcatgtttt tggcacgcgg gatagtgttc atgtgtgtcg aatattgccc 601 gatctttttt atcacaggga atactctgca atgcattatg ctggtttact gtttcttggg 661 ctatttctgc acctgttatt ttggtctatt ctgcctgctt aataggtact ttcgtttgac 721 actaggagtc tatgattact tggtgagcac gcaggagttt cggtatatga acagtcaggg 781 cctgcttccc ccaaagaact ctattgacgc gtttaaactc aatataaagc tgttgggcgt 841 gggtggaaaa ccttgcatca aggtggccac tgtgcaggaa aacctttact ttcagtcgaa 901 gaggcgatgg aaaaagaatt ttatcgcggt gagtgcagcc aacagattca aaaagatatc 961 ctcgtctggt gccctctaag gatccgcagg cctctgctag cttgactgac tgagatacag 1021 cgtaccttca gctcacagac atgataagat acattgatga gtttggacaa accacaacta 1081 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 1141 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 1201 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtattg 1261 gcccatctct atcggtatcg tagcataacc ccttggggcc tctaaacggg tcttgagggg 1321 ttttttgtgc ccctcgggcc ggattgctat ctaccggcat tggcgcagaa aaaaatgcct 1381 gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt 1441 ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct taaggtcgtc 1501 agctatcctg caggcgatct ctcgatttcg atcaagacat tcctttaatg gtcttttctg 1561 gacaccacta ggggtcagaa gtagttcatc aaactttctt ccctccctaa tctcattggt 1621 taccttgggc tatcgaaact taattaacca gtcaagtcag ctacttggcg agatcgactt 1681 gtctgggttt cgactacgct cagaattgcg tcagtcaagt tcgatctggt ccttgctatt 1741 gcacccgttc tccgattacg agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg 1801 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1861 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1921 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1981 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 2041 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 2101 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 2161 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 2221 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 2281 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2341 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2401 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2461 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 2521 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 2581 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 2641 ttcgttcatc catagttgca tttaaatttc cgaactctcc aaggccctcg tcggaaaatc 2701 ttcaaacctt tcgtccgatc catcttgcag gctacctctc gaacgaacta tcgcaagtct 2761 cttggccggc cttgcgcctt ggctattgct tggcagcgcc tatcgccagg tattactcca 2821 atcccgaata tccgagatcg ggatcacccg agagaagttc aacctacatc ctcaatcccg 2881 atctatccga gatccgagga atatcgaaat cggggcgcgc ctggcctccg cgccgggttt 2941 tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc tgccacgtca gacgaagggc 3001 gcaggagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga 3061 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc 3121 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg 3181 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc 3241 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga 3301 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc 3361 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt 3421 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt 3481 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg 3541 ggggcatggt gggcggcaag aacccaaggt cttgagccct tcgctaatgc gggaaagctc 3601 ttattcgggt gagatgggct gggcaccatc tggggaccct gacgtgaagt ttgtcactga 3661 ctggagaact cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg ccgttgggca 3721 gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt 3781 ggcttataat gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg 3841 caggacgcag ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc cggacctctg 3901 gtgaggggag ggataagtga ggcgtcagtt tctttggtcg gttttatgta cctatcttct 3961 taagtagctg aagctccggt tttgaactat gcgctcgggg ttggcgagtg tgttttgtga 4021 agttttttag gcaccttttg aaatgtaatc atttgggtca atatgtaatt ttcagtgtta 4081 gacttgtaaa ttgtccgcta aattctggcc gtttttggct tttttgttag acaacatggg 4141 taaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 4201 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 4261 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 4321 ttatgtttat cggcactttg catccgccgc gctcccgatt ccggaagtgc ttgacattgg 4381 ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 4441 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg caatggatgc 4501 catcgctgcc gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 4561 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 4621 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 4681 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 4741 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 4801 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 4861 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccccg 4921 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 4981 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 5041 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcc gccgtctgga ccgatggctg 5101 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 5161 ataagctagt atgtaagcct agtcttagat aataaaatcg ctatccatcg aagatggatg 5221 tgtgttggtt ttttgtgtgt gtaacgctag gcgcgcctgg tgtaccgaga acgatcctct 5281 cagtgcgagt ctcgacgatc catatcgttg cttggcagtc agccagtcgg aatccagctt 5341 gggacccagg aagtccaatc gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg 5401 atctgtttaa acctagatat tgatagtctg atcggtcaac gtataatcga gtcctagctt 5461 ttgcaaacat ctatcaagag acaggatcag caggaggctt tcgcatgagt attcaacatt 5521 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 5581 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg ggttacatcg 5641 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgctttccaa 5701 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 5761 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag 5821 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 5881 ccatgagtga taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc 5941 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 6001 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 6061 caaccttgcg taaactatta actggcgaac tacttactct agcttcccgg caacagttga 6121 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 6181 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 6241 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 6301 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 6361 ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct 6421 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc 6481 atacgtgtcc tccttaccag aaatttatcc ttaagatcgt ttaaactcga ctctggctct 6541 atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg 6601 catcgaatct cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca 6661 tcttggacca ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca 6721 gctacctctc aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct 6781 atcaatcgtt gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga 6841 ttttattatc taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt 6901 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 6961 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 7021 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 7081 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 7141 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 7201 catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg 7261 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 7321 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 7381 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt 7441 agtgaaccgt cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg 7501 gccgc //