LOCUS Exported 6221 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp11. ACCESSION . VERSION . KEYWORDS pGBW-m4252802 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6221) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6221) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6221 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 41..61 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 65..130 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 183..304 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(194..213) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 248..267 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 419..465 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 495..631 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 676..747 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(973..1561) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1053..1072) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2061..3270 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3107..3126 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3274..3873 /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(3274..3293) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3770..3790 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 3900..3948 /note="synthetic polyadenylation signal" CDS 4214..5074 /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(4432..4451) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5087..5223 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5375..5446 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5451..5497 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5515..5563 /note="synthetic polyadenylation signal" enhancer 5644..5947 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5949..6151 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6102..6122 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 6220..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggct gatgcacaat cgtttttaaa cgggtttgcg gaaaaccttt actttcagtc 61 gatggattat aaggaccacg atggagacta caaagatcat gacattgatt acaaggacga 121 cgatgacaaa taagctagct tgactgactg agatacagcg taccttcagc tcacagacat 181 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 241 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 301 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt 361 tttttaaagc aagtaaaacc tctacaaatg tggtattggc ccatctctat cggtatcgta 421 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc ctcgggccgg 481 attgctatct accggcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat 541 actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata 601 cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag ctatcctgca ggcgatctct 661 cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg ggtcagaagt 721 agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta tcgaaactta 781 attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg actacgctca 841 gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc cgattacgag 901 tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 961 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 1021 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1081 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1141 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 1201 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 1261 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 1321 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1381 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 1441 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 1501 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1561 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1621 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 1681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 1741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcatt 1801 taaatttccg aactctccaa ggccctcgtc ggaaaatctt caaacctttc gtccgatcca 1861 tcttgcaggc tacctctcga acgaactatc gcaagtctct tggccggcct tgcgccttgg 1921 ctattgcttg gcagcgccta tcgccaggta ttactccaat cccgaatatc cgagatcggg 1981 atcacccgag agaagttcaa cctacatcct caatcccgat ctatccgaga tccgaggaat 2041 atcgaaatcg gggcgcgcct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc 2101 cctcgtcacg gcgagcgctg ccacgtcaga cgaagggcgc aggagcgtcc tgatccttcc 2161 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat 2221 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca 2281 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg 2341 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg 2401 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg 2461 agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg 2521 aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa 2581 ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg 2641 cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa 2701 cccaaggtct tgagcccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg 2761 gcaccatctg gggaccctga cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc 2821 tgttgcgggg gcggcagtta tggcggtgcc gttgggcagt gcacccgtac ctttgggagc 2881 gcgcgccctc gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc agggtggggc 2941 caccagccgg taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta 3001 gggtaggctc tcctgaatcg acaggcgccg gacctctggt gaggggaggg ataagtgagg 3061 cgtcagtttc tttggtcggt tttatgtacc tatcttctta agtagctgaa gctccggttt 3121 tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa 3181 atgtaatcat ttgggtcaat atgtaatttt cagtgttaga cttgtaaatt gtccgctaaa 3241 ttctggccgt ttttggcttt tttgttagac aacatgaccg agtacaagcc cacggtgcgc 3301 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 3361 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 3421 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 3481 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 3541 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 3601 gagggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 3661 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 3721 gccgccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 3781 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 3841 acctggtgca tgacccgcaa gcccggtgcc taggctagta tgtaagccta gtcttagata 3901 ataaaatcgc tatccatcga agatggatgt gtgttggttt tttgtgtgtg taacgctagg 3961 cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc 4021 ttggcagtca gccagtcgga atccagcttg ggacccagga agtccaatcg tcagatattg 4081 tactcaagcc tggtcacggc agcgtaccga tctgtttaaa cctagatatt gatagtctga 4141 tcggtcaacg tataatcgag tcctagcttt tgcaaacatc tatcaagaga caggatcagc 4201 aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 4261 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 4321 tcagttgggt gcgcgagtgg gttacatcga actggatctc aacagcggta agatccttga 4381 gagttttcgc cccgaagaac gctttccaat gatgagcact tttaaagttc tgctatgtgg 4441 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 4501 tcagaatgac ttggttgagt attcaccagt cacagaaaag catcttacgg atggcatgac 4561 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 4621 tctgacaacg attggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 4681 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 4741 tgacaccacg atgcctgtag caatggcaac aaccttgcgt aaactattaa ctggcgaact 4801 acttactcta gcttcccggc aacagttgat agactggatg gaggcggata aagttgcagg 4861 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 4921 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 4981 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 5041 tgagataggt gcctcactga ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa 5101 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg 5161 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct 5221 taagatcccg aatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct 5281 tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc 5341 agcttacctt tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt 5401 atcttcttcc ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc 5461 ctcaagaccc gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca 5521 aaaaaccaac acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga 5581 gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat atatggagtt 5641 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 5701 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 5761 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 5821 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 5881 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 5941 taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg 6001 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca 6061 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg 6121 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt 6181 gtcgatccta ccatccactc gacacacccg ccagcggccg c //