LOCUS Exported 6935 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp9. ACCESSION . VERSION . KEYWORDS pGBW-m4133825 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6935) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6935) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6935 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 341..361 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 362..406 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" polyA_signal 475..596 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(486..505) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 540..559 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 711..757 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 787..923 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 968..1039 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1265..1853) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1345..1364) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2353..3562 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3399..3418 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3566..4594 /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 4621..4669 /note="synthetic polyadenylation signal" CDS 4935..5795 /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(5153..5172) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5808..5944 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6089..6160 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6165..6211 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6229..6277 /note="synthetic polyadenylation signal" enhancer 6358..6661 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6663..6865 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6816..6836 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgaat gagcttagtc ctgttgcact acgacagatg tcttgtgctg ccggtactac 61 acaaactgct tgcactgatg acaatgcgtt agcttactac aacacaacaa agggaggtag 121 gtttgtactt gcactgttat ccgatttaca ggatttgaaa tgggctagat tccctaagag 181 tgatggaact ggtactatct atacagaact ggaaccacct tgtaggtttg ttacagacac 241 acctaaaggt cctaaagtga agtatttata ctttattaaa ggattaaaca acctaaatag 301 aggtatggta cttggtagtt tagctgccac agtacgtcta gaaaaccttt actttcagtc 361 gaaagaaacc gcggcagcta agttcgagag gcagcacatg gacagttaag gatccgcagg 421 cctctgctag cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat 481 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg 541 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca 601 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa 661 gcaagtaaaa cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc 721 ccttggggcc tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat 781 ctaccggcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac 841 atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct 901 ccttaccaga aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg 961 atcaagacat tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc 1021 aaactttctt ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca 1081 gtcaagtcag ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg 1141 tcagtcaagt tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt 1201 aaatcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 1261 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 1321 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 1381 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 1441 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 1501 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 1561 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 1621 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 1681 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 1741 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 1801 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 1861 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 1921 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 1981 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 2041 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc 2101 cgaactctcc aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag 2161 gctacctctc gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct 2221 tggcagcgcc tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg 2281 agagaagttc aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat 2341 cggggcgcgc ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca 2401 cggcgagcgc tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac 2461 gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa 2521 ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg 2581 aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg 2641 tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc 2701 gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg 2761 gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt 2821 ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt 2881 tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag 2941 gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt 3001 cttgagccct tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc 3061 tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg 3121 gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc 3181 tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc 3241 ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc 3301 tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt 3361 tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat 3421 gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc 3481 atttgggtca atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc 3541 gtttttggct tttttgttag acaacatggg taaaaagcct gaactcaccg cgacgtctgt 3601 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 3661 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 3721 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catccgccgc 3781 gctcccgatt ccggaagtgc ttgacattgg ggagttcagc gagagcctga cctattgcat 3841 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 3901 tctgcagccg gtcgcggagg caatggatgc catcgctgcc gccgatctta gccagacgag 3961 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 4021 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 4081 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 4141 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 4201 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 4261 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 4321 gaggcatccg gagcttgcag gatcgccccg gctccgggcg tatatgctcc gcattggtct 4381 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 4441 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 4501 cagaagcgcc gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 4561 acgccccagc actcgtccga gggcaaagga ataagctagt atgtaagcct agtcttagat 4621 aataaaatcg ctatccatcg aagatggatg tgtgttggtt ttttgtgtgt gtaacgctag 4681 gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg 4741 cttggcagtc agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt 4801 gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg 4861 atcggtcaac gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag 4921 caggaggctt tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 4981 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 5041 atcagttggg tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg 5101 agagttttcg ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg 5161 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 5221 ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga 5281 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 5341 ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 5401 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 5461 gtgacaccac gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac 5521 tacttactct agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag 5581 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 5641 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 5701 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 5761 ctgagatagg tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga 5821 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac 5881 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc 5941 ttaagatcgt ttaaactcga ctctggctct atcgaatctc cgtcgtttcg agcttacgcg 6001 aacagccgtg gcgctcattt gctcgtcggg catcgaatct cgtcagctat cgtcagctta 6061 cctttttggc agcgatcgcg gctcccgaca tcttggacca ttagctccac aggtatcttc 6121 ttccctctag tggtcataac agcagcttca gctacctctc aattcaaaaa acccctcaag 6181 acccgtttag aggccccaag gggttatgct atcaatcgtt gcgttacaca cacaaaaaac 6241 caacacacat ccatcttcga tggatagcga ttttattatc taactgctga tcgagtgtag 6301 ccagatctag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 6361 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 6421 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 6481 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 6541 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 6601 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 6661 gctgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt 6721 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 6781 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg 6841 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcagat ctttgtcgat 6901 cctaccatcc actcgacaca cccgccagcg gccgc //