LOCUS Exported 7362 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp7. ACCESSION . VERSION . KEYWORDS pGBW-m4252889 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7362) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7362) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7362 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 33..52 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 204..250 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 280..416 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 461..532 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(758..1346) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(838..857) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 1846..3055 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 2892..2911 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3059..4087 /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 4114..4162 /note="synthetic polyadenylation signal" CDS 4428..5288 /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(4646..4665) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5301..5437 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5589..5660 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5665..5711 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5729..5777 /note="synthetic polyadenylation signal" enhancer 5858..6161 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6163..6365 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6316..6336 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 6686..6706 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 6740..6817 /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" CDS 6845..6865 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 7330..89 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7341..7360) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg 61 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 121 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta 181 ttggcccatc tctatcggta tcgtagcata accccttggg gcctctaaac gggtcttgag 241 gggttttttg tgcccctcgg gccggattgc tatctaccgg cattggcgca gaaaaaaatg 301 cctgatgcga cgctgcgcgt cttatactcc cacatatgcc agattcagca acggatacgg 361 cttccccaac ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaaggtc 421 gtcagctatc ctgcaggcga tctctcgatt tcgatcaaga cattccttta atggtctttt 481 ctggacacca ctaggggtca gaagtagttc atcaaacttt cttccctccc taatctcatt 541 ggttaccttg ggctatcgaa acttaattaa ccagtcaagt cagctacttg gcgagatcga 601 cttgtctggg tttcgactac gctcagaatt gcgtcagtca agttcgatct ggtccttgct 661 attgcacccg ttctccgatt acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa 721 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 781 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 841 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 901 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 961 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 1021 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 1081 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 1141 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 1201 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 1261 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 1321 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 1381 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 1441 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 1501 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1561 tatttcgttc atccatagtt gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa 1621 atcttcaaac ctttcgtccg atccatcttg caggctacct ctcgaacgaa ctatcgcaag 1681 tctcttggcc ggccttgcgc cttggctatt gcttggcagc gcctatcgcc aggtattact 1741 ccaatcccga atatccgaga tcgggatcac ccgagagaag ttcaacctac atcctcaatc 1801 ccgatctatc cgagatccga ggaatatcga aatcggggcg cgcctggcct ccgcgccggg 1861 ttttggcgcc tcccgcgggc gcccccctcg tcacggcgag cgctgccacg tcagacgaag 1921 ggcgcaggag cgtcctgatc cttccgcccg gacgctcagg acagcggccc gctgctcata 1981 agactcggcc ttagaacccc agtatcagca gaaggacatt ttaggacggg acttgggtga 2041 ctctagggca ctggttttct ttccagagag cggaacaggc gaggaaaagt agtcccttct 2101 cggcgattct gcggagggat ctccgtgggg cggtgaacgc cgatgattat ataaggacgc 2161 gccgggtgtg gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt 2221 ggatcgctgt gatcgtcact tggtgagtag cgggctgctg ggctggccgg ggctttcgtg 2281 gccgccgggc cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg 2341 ggtccgcgag caaggttgcc ctgaactggg ggttgggggg agcgcagcaa aatggcggct 2401 gttcccgagt cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg 2461 tggggggcat ggtgggcggc aagaacccaa ggtcttgagc ccttcgctaa tgcgggaaag 2521 ctcttattcg ggtgagatgg gctgggcacc atctggggac cctgacgtga agtttgtcac 2581 tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg gtgccgttgg 2641 gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct 2701 gttggcttat aatgcagggt ggggccacca gccggtaggt gtgcggtagg cttttctccg 2761 tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct 2821 ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct 2881 tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg 2941 tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg 3001 ttagacttgt aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacaacat 3061 gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga 3121 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga 3181 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga 3241 tcgttatgtt tatcggcact ttgcatccgc cgcgctcccg attccggaag tgcttgacat 3301 tggggagttc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt 3361 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggcaatgga 3421 tgccatcgct gccgccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg 3481 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta 3541 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga 3601 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg 3661 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc 3721 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc 3781 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc 3841 ccggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga 3901 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg 3961 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gccgccgtct ggaccgatgg 4021 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa 4081 ggaataagct agtatgtaag cctagtctta gataataaaa tcgctatcca tcgaagatgg 4141 atgtgtgttg gttttttgtg tgtgtaacgc taggcgcgcc tggtgtaccg agaacgatcc 4201 tctcagtgcg agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag 4261 cttgggaccc aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta 4321 ccgatctgtt taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag 4381 cttttgcaaa catctatcaa gagacaggat cagcaggagg ctttcgcatg agtattcaac 4441 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 4501 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcgcga gtgggttaca 4561 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc 4621 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 4681 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac 4741 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 4801 taaccatgag tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg 4861 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 4921 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 4981 caacaacctt gcgtaaacta ttaactggcg aactacttac tctagcttcc cggcaacagt 5041 tgatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5101 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5161 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5221 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5281 attggtaacc gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg 5341 tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact 5401 tccatacgtg tcctccttac cagaaattta tccttaagat cccgaatcgt ttaaactcga 5461 ctctggctct atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt 5521 gctcgtcggg catcgaatct cgtcagctat cgtcagctta cctttttggc agcgatcgcg 5581 gctcccgaca tcttggacca ttagctccac aggtatcttc ttccctctag tggtcataac 5641 agcagcttca gctacctctc aattcaaaaa acccctcaag acccgtttag aggccccaag 5701 gggttatgct atcaatcgtt gcgttacaca cacaaaaaac caacacacat ccatcttcga 5761 tggatagcga ttttattatc taactgctga tcgagtgtag ccagatctag taatcaatta 5821 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 5881 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 5941 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 6001 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 6061 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 6121 cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt 6181 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 6241 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 6301 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 6361 gagctggttt agtgaaccgt cagatcagat ctttgtcgat cctaccatcc actcgacaca 6421 cccgccagcg gccgccacca tgaaaatgtc agatgtaaag tgcacatcag tagtcttact 6481 ctcagttttg caacaactca gagtagaatc atcatctaaa ttgtgggctc aatgtgtcca 6541 gttacacaat gacattctct tagctaaaga tactactgaa gcctttgaaa aaatggtttc 6601 actactttct gttttgcttt ccatgcaggg tgctgtagac ataaacaagc tttgtgaaga 6661 aatgctggac aacagggcaa ccttagaaaa cctttacttt cagtcgggac ggcgaatccc 6721 ggggctgata aatccctgga aaaggagatg gaagaagaac tttattgcag tgtctgcggc 6781 taaccggttc aaaaagatca gttcaagcgg cgccctagat tacgacattc ctacgaccgc 6841 ctccgagaat ctctatttcc agggtgaatt tggcttggca caacatgacg aggccgtcga 6901 taacaagttt aataaagaac agcagaacgc tttctatgag atccttcact taccaaatct 6961 gaacgaagag caacgcaacg ctttcattca gagcctgaaa gacgatccct cgcagtctgc 7021 aaatctcctg gccgaagcca agaagctgaa tgacgcacag gctccaaaag ttgataacaa 7081 gtttaacaag gagcagcaga atgctttcta cgagatattg caccttccta atctgaatga 7141 ggaacaaaga aatgcattta tccagtccct aaaagacgat ccatcacaga gtgccaacct 7201 gcttgcggaa gccaaaaagc tcaatgatgc ccaggccccg aaagtagacg cgaaccatca 7261 ataaggatcc gcaggcctct gctagcttga ctgactgaga tacagcgtac cttcagctca 7321 cagacatgat aagatacatt gatgagtttg gacaaaccac aa //