LOCUS Exported 6994 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp6. ACCESSION . VERSION . KEYWORDS pGBW-m4134399 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6994) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6994) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6994 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 872..892 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 893..910 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 963..1084 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(974..993) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1028..1047 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1199..1245 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1275..1411 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1456..1527 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1753..2341) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1833..1852) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2841..4050 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3887..3906 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4054..4653 /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(4054..4073) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4550..4570 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4680..4728 /note="synthetic polyadenylation signal" CDS 4994..5854 /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(5212..5231) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5867..6003 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6148..6219 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6224..6270 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6288..6336 /note="synthetic polyadenylation signal" enhancer 6417..6720 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6722..6924 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6875..6895 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 6993..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggca gtgaaaagaa caatcaaggg tacacaccac tggttgttac tcacaatttt 61 gacttcactt ttagttttag tccagagtac tcaatggtct ttgttctttt ttttgtatga 121 aaatgccttt ttaccttttg ctatgggtat tattgctatg tctgcttttg caatgatgtt 181 tgtcaaacat aagcatgcat ttctctgttt gtttttgtta ccttctcttg ccactgtagc 241 ttattttaat atggtctata tgcctgctag ttgggtgatg cgtattatga catggttgga 301 tatggttgat actagtttgt ctggttttaa gctaaaagac tgtgttatgt atgcatcagc 361 tgtagtgtta ctaatcctta tgacagcaag aactgtgtat gatgatggtg ctaggagagt 421 gtggacactt atgaatgtct tgacactcgt ttataaagtt tattatggta atgctttaga 481 tcaagccatt tccatgtggg ctcttataat ctctgttact tctaactact caggtgtagt 541 tacaactgtc atgtttttgg ccagaggtat tgtttttatg tgtgttgagt attgccctat 601 tttcttcata actggtaata cacttcagtg tataatgcta gtttattgtt tcttaggcta 661 tttttgtact tgttactttg gcctcttttg tttactcaac cgctacttta gactgactct 721 tggtgtttat gattacttag tttctacaca ggagtttaga tatatgaatt cacagggact 781 actcccaccc aagaatagca tagatgcctt caaactcaac attaaattgt tgggtgttgg 841 tggcaaacct tgtatcaaag tagccactgt agaaaacctt tactttcagt cgcaccatca 901 ccatcaccat taagctagct tgactgactg agatacagcg taccttcagc tcacagacat 961 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 1021 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 1081 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt 1141 tttttaaagc aagtaaaacc tctacaaatg tggtattggc ccatctctat cggtatcgta 1201 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc ctcgggccgg 1261 attgctatct accggcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat 1321 actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata 1381 cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag ctatcctgca ggcgatctct 1441 cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg ggtcagaagt 1501 agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta tcgaaactta 1561 attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg actacgctca 1621 gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc cgattacgag 1681 tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 1741 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 1801 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1861 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1921 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 1981 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2041 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2101 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2161 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 2221 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2281 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2341 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2401 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 2461 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 2521 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcatt 2581 taaatttccg aactctccaa ggccctcgtc ggaaaatctt caaacctttc gtccgatcca 2641 tcttgcaggc tacctctcga acgaactatc gcaagtctct tggccggcct tgcgccttgg 2701 ctattgcttg gcagcgccta tcgccaggta ttactccaat cccgaatatc cgagatcggg 2761 atcacccgag agaagttcaa cctacatcct caatcccgat ctatccgaga tccgaggaat 2821 atcgaaatcg gggcgcgcct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc 2881 cctcgtcacg gcgagcgctg ccacgtcaga cgaagggcgc aggagcgtcc tgatccttcc 2941 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat 3001 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca 3061 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg 3121 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg 3181 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg 3241 agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg 3301 aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa 3361 ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg 3421 cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa 3481 cccaaggtct tgagcccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg 3541 gcaccatctg gggaccctga cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc 3601 tgttgcgggg gcggcagtta tggcggtgcc gttgggcagt gcacccgtac ctttgggagc 3661 gcgcgccctc gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc agggtggggc 3721 cacctgccgg taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta 3781 gggtaggctc tcctgaatcg acaggcgccg gacctctggt gaggggaggg ataagtgagg 3841 cgtcagtttc tttggtcggt tttatgtacc tatcttctta agtagctgaa gctccggttt 3901 tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa 3961 atgtaatcat ttgggtcaat atgtaatttt cagtgttaga cttgtaaatt gtccgctaaa 4021 ttctggccgt ttttggcttt tttgttagac aacatgaccg agtacaagcc cacggtgcgc 4081 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 4141 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 4201 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 4261 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 4321 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 4381 gagggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 4441 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 4501 gccgccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 4561 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 4621 acctggtgca tgacccgcaa gcccggtgcc taggctagta tgtaagccta gtcttagata 4681 ataaaatcgc tatccatcga agatggatgt gtgttggttt tttgtgtgtg taacgctagg 4741 cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc 4801 ttggcagtca gccagtcgga atccagcttg ggacccagga agtccaatcg tcagatattg 4861 tactcaagcc tggtcacggc agcgtaccga tctgtttaaa cctagatatt gatagtctga 4921 tcggtcaacg tataatcgag tcctagcttt tgcaaacatc tatcaagaga caggatcagc 4981 aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 5041 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 5101 tcagttgggt gcgcgagtgg gttacatcga actggatctc aacagcggta agatccttga 5161 gagttttcgc cccgaagaac gctttccaat gatgagcact tttaaagttc tgctatgtgg 5221 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 5281 tcagaatgac ttggttgagt attcaccagt cacagaaaag catcttacgg atggcatgac 5341 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 5401 tctgacaacg attggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 5461 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 5521 tgacaccacg atgcctgtag caatggcaac aaccttgcgt aaactattaa ctggcgaact 5581 acttactcta gcttcccggc aacagttgat agactggatg gaggcggata aagttgcagg 5641 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 5701 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 5761 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 5821 tgagataggt gcctcactga ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa 5881 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg 5941 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct 6001 taagatcgtt taaactcgac tctggctcta tcgaatctcc gtcgtttcga gcttacgcga 6061 acagccgtgg cgctcatttg ctcgtcgggc atcgaatctc gtcagctatc gtcagcttac 6121 ctttttggca gcgatcgcgg ctcccgacat cttggaccat tagctccaca ggtatcttct 6181 tccctctagt ggtcataaca gcagcttcag ctacctctca attcaaaaaa cccctcaaga 6241 cccgtttaga ggccccaagg ggttatgcta tcaatcgttg cgttacacac acaaaaaacc 6301 aacacacatc catcttcgat ggatagcgat tttattatct aactgctgat cgagtgtagc 6361 cagatctagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 6421 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 6481 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 6541 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 6601 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 6661 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 6721 ctgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 6781 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 6841 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 6901 tgggaggtct atataagcag agctggttta gtgaaccgtc agatcagatc tttgtcgatc 6961 ctaccatcca ctcgacacac ccgccagcgg ccgc //