LOCUS Exported 7021 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp6. ACCESSION . VERSION . KEYWORDS pGBW-m4133237 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7021) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7021) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7021 /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..937 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" polyA_signal 990..1111 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1001..1020) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1055..1074 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1226..1272 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1302..1438 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1483..1554 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1780..2368) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1860..1879) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2868..4077 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3914..3933 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4081..4680 /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(4081..4100) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4577..4597 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4707..4755 /note="synthetic polyadenylation signal" CDS 5021..5881 /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(5239..5258) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5894..6030 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6175..6246 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6251..6297 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6315..6363 /note="synthetic polyadenylation signal" enhancer 6444..6747 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6749..6951 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6902..6922 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7020..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 cgaaagaaac 901 cgcggcagct aagttcgaga ggcagcacat ggacagttaa gctagcttga ctgactgaga 961 tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg gacaaaccac 1021 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 1081 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 1141 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 1201 tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa acgggtcttg 1261 aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg cagaaaaaaa 1321 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac 1381 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaagg 1441 tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt taatggtctt 1501 ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc cctaatctca 1561 ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact tggcgagatc 1621 gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat ctggtccttg 1681 ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca 1741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 1801 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 1861 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 1921 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 1981 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 2041 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 2101 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 2161 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 2221 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 2281 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 2341 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 2401 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 2461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 2521 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 2581 tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga 2641 aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca 2701 agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta 2761 ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa 2821 tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg 2881 ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca cgtcagacga 2941 agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc ccgctgctca 3001 taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg ggacttgggt 3061 gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa gtagtccctt 3121 ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt atataaggac 3181 gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg gttcttgttt 3241 gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc ggggctttcg 3301 tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag ggctgtagtc 3361 tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg 3421 ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa 3481 ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa 3541 agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt gaagtttgtc 3601 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt 3661 gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt 3721 ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc 3781 cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac 3841 ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat 3901 cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt 3961 tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag 4021 tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacaac 4081 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 4141 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 4201 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 4261 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 4321 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 4381 tcccggctgg ccgcgcagca acagatggag ggcctcctgg cgccgcaccg gcccaaggag 4441 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 4501 agcgccgtcg tgctccccgg agtggaggcc gccgagcgcg ccggggtgcc cgccttcctg 4561 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 4621 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctag 4681 gctagtatgt aagcctagtc ttagataata aaatcgctat ccatcgaaga tggatgtgtg 4741 ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga tcctctcagt 4801 gcgagtctcg acgatccata tcgttgcttg gcagtcagcc agtcggaatc cagcttggga 4861 cccaggaagt ccaatcgtca gatattgtac tcaagcctgg tcacggcagc gtaccgatct 4921 gtttaaacct agatattgat agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc 4981 aaacatctat caagagacag gatcagcagg aggctttcgc atgagtattc aacatttccg 5041 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5101 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt acatcgaact 5161 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgct ttccaatgat 5221 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5281 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtatt caccagtcac 5341 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5401 gagtgataac actgcggcca acttacttct gacaacgatt ggaggaccga aggagctaac 5461 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5521 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5581 cttgcgtaaa ctattaactg gcgaactact tactctagct tcccggcaac agttgataga 5641 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5701 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5761 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5821 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5881 accgattcta ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata 5941 ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc acttccatac 6001 gtgtcctcct taccagaaat ttatccttaa gatcgtttaa actcgactct ggctctatcg 6061 aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc 6121 gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc ccgacatctt 6181 ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca gcttcagcta 6241 cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctatca 6301 atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga tagcgatttt 6361 attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt 6421 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 6481 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 6541 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 6601 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 6661 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 6721 tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacat caatgggcgt 6781 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 6841 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 6901 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg 6961 aaccgtcaga tcagatcttt gtcgatccta ccatccactc gacacacccg ccagcggccg 7021 c //