LOCUS Exported 7361 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp7. ACCESSION . VERSION . KEYWORDS pGBW-m4133243 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7361) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7361) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7361 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 257..277 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 311..388 /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 416..436 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 901..1022 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(912..931) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 966..985 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1137..1183 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1213..1349 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1394..1465 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1691..2279) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1771..1790) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2779..3988 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3825..3844 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3992..5020 /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 5047..5095 /note="synthetic polyadenylation signal" CDS 5361..6221 /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(5579..5598) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6234..6370 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6515..6586 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6591..6637 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6655..6703 /note="synthetic polyadenylation signal" enhancer 6784..7087 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7089..7291 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7242..7262 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgagc aaaatgtctg acgtcaagtg tacatccgta gttctgttat cagtgcttca 61 acagctgcgg gtggagagtt cgtcaaagct ctgggcccag tgcgtgcagc tgcacaatga 121 tatattgctc gcgaaagata ctacggaagc tttcgagaag atggtcagct tgctatctgt 181 tctactgagc atgcaaggag cagtagacat caacaaactt tgcgaagaga tgttagacaa 241 ccgagccacc ctgcaagaaa acctttactt tcagtcggga cggcgaatcc cggggctgat 301 aaatccctgg aaaaggagat ggaagaagaa ctttattgca gtgtctgcgg ctaaccggtt 361 caaaaagatc agttcaagcg gcgccctaga ttacgacatt cctacgaccg cctccgagaa 421 tctctatttc cagggtgaat ttggcttggc acaacatgac gaggccgtcg ataacaagtt 481 taataaagaa cagcagaacg ctttctatga gatccttcac ttaccaaatc tgaacgaaga 541 gcaacgcaac gctttcattc agagcctgaa agacgatccc tcgcagtctg caaatctcct 601 ggccgaagcc aagaagctga atgacgcaca ggctccaaaa gttgataaca agtttaacaa 661 ggagcagcag aatgctttct acgagatatt gcaccttcct aatctgaatg aggaacaaag 721 aaatgcattt atccagtccc taaaagacga tccatcacag agtgccaacc tgcttgcgga 781 agccaaaaag ctcaatgatg cccaggcccc gaaagtagac gcgaaccatc aataaggatc 841 cgcaggcctc tgctagcttg actgactgag atacagcgta ccttcagctc acagacatga 901 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 961 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 1021 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt 1081 tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc atctctatcg gtatcgtagc 1141 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgtgcccct cgggccggat 1201 tgctatctac cggcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac 1261 tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg 1321 tgtcctcctt accagaaatt tatccttaag gtcgtcagct atcctgcagg cgatctctcg 1381 atttcgatca agacattcct ttaatggtct tttctggaca ccactagggg tcagaagtag 1441 ttcatcaaac tttcttccct ccctaatctc attggttacc ttgggctatc gaaacttaat 1501 taaccagtca agtcagctac ttggcgagat cgacttgtct gggtttcgac tacgctcaga 1561 attgcgtcag tcaagttcga tctggtcctt gctattgcac ccgttctccg attacgagtt 1621 tcatttaaat catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 1681 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 1741 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 1801 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 1861 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 1921 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 1981 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 2041 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 2101 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 2161 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 2221 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 2281 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 2341 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 2401 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 2461 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta 2521 aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc 2581 ttgcaggcta cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct 2641 attgcttggc agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat 2701 cacccgagag aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat 2761 cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc 2821 tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag gagcgtcctg atccttccgc 2881 ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca 2941 gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt tctttccaga 3001 gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg 3061 gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc 3121 gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag 3181 tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa 3241 gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact 3301 gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct 3361 tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc 3421 caaggtcttg agcccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggc 3481 accatctggg gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg 3541 ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc 3601 gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca 3661 cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg 3721 gtaggctctc ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg 3781 tcagtttctt tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg 3841 aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat 3901 gtaatcattt gggtcaatat gtaattttca gtgttagact tgtaaattgt ccgctaaatt 3961 ctggccgttt ttggcttttt tgttagacaa catgggtaaa aagcctgaac tcaccgcgac 4021 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 4081 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 4141 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 4201 cgccgcgctc ccgattccgg aagtgcttga cattggggag ttcagcgaga gcctgaccta 4261 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 4321 cgctgttctg cagccggtcg cggaggcaat ggatgccatc gctgccgccg atcttagcca 4381 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 4441 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 4501 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 4561 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 4621 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 4681 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 4741 cgagcggagg catccggagc ttgcaggatc gccccggctc cgggcgtata tgctccgcat 4801 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 4861 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 4921 cgcccgcaga agcgccgccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 4981 aaaccgacgc cccagcactc gtccgagggc aaaggaataa gctagtatgt aagcctagtc 5041 ttagataata aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa 5101 cgctaggcgc gcctggtgta ccgagaacga tcctctcagt gcgagtctcg acgatccata 5161 tcgttgcttg gcagtcagcc agtcggaatc cagcttggga cccaggaagt ccaatcgtca 5221 gatattgtac tcaagcctgg tcacggcagc gtaccgatct gtttaaacct agatattgat 5281 agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc aaacatctat caagagacag 5341 gatcagcagg aggctttcgc atgagtattc aacatttccg tgtcgccctt attccctttt 5401 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 5461 ctgaagatca gttgggtgcg cgagtgggtt acatcgaact ggatctcaac agcggtaaga 5521 tccttgagag ttttcgcccc gaagaacgct ttccaatgat gagcactttt aaagttctgc 5581 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 5641 actattctca gaatgacttg gttgagtatt caccagtcac agaaaagcat cttacggatg 5701 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 5761 acttacttct gacaacgatt ggaggaccga aggagctaac cgcttttttg cacaacatgg 5821 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 5881 acgagcgtga caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg 5941 gcgaactact tactctagct tcccggcaac agttgataga ctggatggag gcggataaag 6001 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 6061 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 6121 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 6181 agatcgctga gataggtgcc tcactgatta agcattggta accgattcta ggtgcattgg 6241 cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc 6301 agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat 6361 ttatccttaa gatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct 6421 tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc 6481 agcttacctt tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt 6541 atcttcttcc ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc 6601 ctcaagaccc gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca 6661 aaaaaccaac acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga 6721 gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat atatggagtt 6781 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 6841 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 6901 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 6961 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 7021 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 7081 taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg 7141 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca 7201 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg 7261 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt 7321 gtcgatccta ccatccactc gacacacccg ccagcggccg c //