LOCUS Exported 6431 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp7. ACCESSION . VERSION . KEYWORDS pGBW-m4252839 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6431) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6431) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6431 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 251..271 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 275..340 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 393..514 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(404..423) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 458..477 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 629..675 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 705..841 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 886..957 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1183..1771) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1263..1282) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2271..3480 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3317..3336 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3484..4083 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(3484..3503) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3980..4000 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4110..4158 /note="synthetic polyadenylation signal" CDS 4424..5284 /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(4642..4661) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5297..5433 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5585..5656 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5661..5707 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5725..5773 /note="synthetic polyadenylation signal" enhancer 5854..6157 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6159..6361 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6312..6332 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgaaa atgtcagatg taaagtgcac atcagtagtc ttactctcag ttttgcaaca 61 actcagagta gaatcatcat ctaaattgtg ggctcaatgt gtccagttac acaatgacat 121 tctcttagct aaagatacta ctgaagcctt tgaaaaaatg gtttcactac tttctgtttt 181 gctttccatg cagggtgctg tagacataaa caagctttgt gaagaaatgc tggacaacag 241 ggcaacctta gaaaaccttt actttcagtc gatggattat aaggaccacg atggagacta 301 caaagatcat gacattgatt acaaggacga cgatgacaaa taagctagct tgactgactg 361 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac 421 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 481 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 541 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 601 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc 661 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa 721 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga 781 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta 841 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt 901 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc 961 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag 1021 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc 1081 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca 1141 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 1201 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 1261 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 1321 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 1381 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 1441 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 1501 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 1561 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 1621 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 1681 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 1741 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 1801 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 1861 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 1921 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 1981 ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc 2041 ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc 2101 gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta 2161 ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct 2221 caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg 2281 ccgggttttg gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga 2341 cgaagggcgc aggagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc 2401 tcataagact cggccttaga accccagtat cagcagaagg acattttagg acgggacttg 2461 ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc 2521 cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag 2581 gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg 2641 tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt 2701 tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta 2761 gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg 2821 cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa 2881 caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg 2941 gaaagctctt attcgggtga gatgggctgg gcaccatctg gggaccctga cgtgaagttt 3001 gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc 3061 gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc 3121 gttctgttgg cttataatgc agggtggggc caccagccgg taggtgtgcg gtaggctttt 3181 ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg acaggcgccg 3241 gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt tttatgtacc 3301 tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt ggcgagtgtg 3361 ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat atgtaatttt 3421 cagtgttaga cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt tttgttagac 3481 aacatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc 3541 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg 3601 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc 3661 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg 3721 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc 3781 ggttcccggc tggccgcgca gcaacagatg gagggcctcc tggcgccgca ccggcccaag 3841 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg 3901 ggcagcgccg tcgtgctccc cggagtggag gccgccgagc gcgccggggt gcccgccttc 3961 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc 4021 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc 4081 taggctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt 4141 gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc 4201 agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg 4261 ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga 4321 tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt 4381 tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt 4441 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 4501 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga 4561 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat 4621 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 4681 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt 4741 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 4801 catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct 4861 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 4921 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 4981 aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat 5041 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 5101 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 5161 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 5221 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5281 gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt 5341 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca 5401 tacgtgtcct ccttaccaga aatttatcct taagatcccg aatcgtttaa actcgactct 5461 ggctctatcg aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc 5521 gtcgggcatc gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc 5581 ccgacatctt ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca 5641 gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt 5701 tatgctatca atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga 5761 tagcgatttt attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg 5821 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 5881 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 5941 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 6001 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 6061 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 6121 gcagtacatc tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacat 6181 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 6241 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 6301 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 6361 tggtttagtg aaccgtcaga tcagatcttt gtcgatccta ccatccactc gacacacccg 6421 ccagcggccg c //