LOCUS Exported 7490 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp6. ACCESSION . VERSION . KEYWORDS pGBW-m4134260 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7490) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7490) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7490 /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 896..961 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 1030..1151 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1041..1060) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1095..1114 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1266..1312 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1342..1478 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1523..1594 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1820..2408) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1900..1919) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2908..4117 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3954..3973 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4121..5149 /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 5176..5224 /note="synthetic polyadenylation signal" CDS 5490..6350 /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(5708..5727) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6363..6499 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6644..6715 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6720..6766 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6784..6832 /note="synthetic polyadenylation signal" enhancer 6913..7216 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7218..7420 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7371..7391 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7489..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 cgatggatta 901 taaggaccac gatggagact acaaagatca tgacattgat tacaaggacg acgatgacaa 961 ataaggatcc gcaggcctct gctagcttga ctgactgaga tacagcgtac cttcagctca 1021 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 1081 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1141 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1201 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tattggccca tctctatcgg 1261 tatcgtagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgtgcccctc 1321 gggccggatt gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc 1381 gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac 1441 ttccatacgt gtcctcctta ccagaaattt atccttaagg tcgtcagcta tcctgcaggc 1501 gatctctcga tttcgatcaa gacattcctt taatggtctt ttctggacac cactaggggt 1561 cagaagtagt tcatcaaact ttcttccctc cctaatctca ttggttacct tgggctatcg 1621 aaacttaatt aaccagtcaa gtcagctact tggcgagatc gacttgtctg ggtttcgact 1681 acgctcagaa ttgcgtcagt caagttcgat ctggtccttg ctattgcacc cgttctccga 1741 ttacgagttt catttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 1801 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 1861 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 1921 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 1981 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 2041 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 2101 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 2161 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 2221 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 2281 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 2341 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 2401 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 2461 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 2521 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 2581 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 2641 ttgcatttaa atttccgaac tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc 2701 cgatccatct tgcaggctac ctctcgaacg aactatcgca agtctcttgg ccggccttgc 2761 gccttggcta ttgcttggca gcgcctatcg ccaggtatta ctccaatccc gaatatccga 2821 gatcgggatc acccgagaga agttcaacct acatcctcaa tcccgatcta tccgagatcc 2881 gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg 2941 gcgcccccct cgtcacggcg agcgctgcca cgtcagacga agggcgcagg agcgtcctga 3001 tccttccgcc cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc 3061 ccagtatcag cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt 3121 ctttccagag agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg 3181 atctccgtgg ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct 3241 agttccgtcg cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca 3301 cttggtgagt agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt 3361 gggacggaag cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg 3421 ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg 3481 gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg 3541 gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa agctcttatt cgggtgagat 3601 gggctgggca ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt 3661 tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt 3721 tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg 3781 gtggggccac ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc 3841 gggcctaggg taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata 3901 agtgaggcgt cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct 3961 ccggttttga actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc 4021 ttttgaaatg taatcatttg ggtcaatatg taattttcag tgttagactt gtaaattgtc 4081 cgctaaattc tggccgtttt tggctttttt gttagacaac atgggtaaaa agcctgaact 4141 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat 4201 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata 4261 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca 4321 ctttgcatcc gccgcgctcc cgattccgga agtgcttgac attggggagt tcagcgagag 4381 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac 4441 cgaactgccc gctgttctgc agccggtcgc ggaggcaatg gatgccatcg ctgccgccga 4501 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac 4561 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat 4621 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga 4681 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac 4741 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca 4801 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac 4861 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccccggctcc gggcgtatat 4921 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc 4981 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg 5041 tacacaaatc gcccgcagaa gcgccgccgt ctggaccgat ggctgtgtag aagtactcgc 5101 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataag ctagtatgta 5161 agcctagtct tagataataa aatcgctatc catcgaagat ggatgtgtgt tggttttttg 5221 tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat cctctcagtg cgagtctcga 5281 cgatccatat cgttgcttgg cagtcagcca gtcggaatcc agcttgggac ccaggaagtc 5341 caatcgtcag atattgtact caagcctggt cacggcagcg taccgatctg tttaaaccta 5401 gatattgata gtctgatcgg tcaacgtata atcgagtcct agcttttgca aacatctatc 5461 aagagacagg atcagcagga ggctttcgca tgagtattca acatttccgt gtcgccctta 5521 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 5581 taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta catcgaactg gatctcaaca 5641 gcggtaagat ccttgagagt tttcgccccg aagaacgctt tccaatgatg agcactttta 5701 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 5761 gccgcataca ctattctcag aatgacttgg ttgagtattc accagtcaca gaaaagcatc 5821 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 5881 ctgcggccaa cttacttctg acaacgattg gaggaccgaa ggagctaacc gcttttttgc 5941 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 6001 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacc ttgcgtaaac 6061 tattaactgg cgaactactt actctagctt cccggcaaca gttgatagac tggatggagg 6121 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 6181 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 6241 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 6301 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ccgattctag 6361 gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat 6421 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt 6481 accagaaatt tatccttaag atcgtttaaa ctcgactctg gctctatcga atctccgtcg 6541 tttcgagctt acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca 6601 gctatcgtca gcttaccttt ttggcagcga tcgcggctcc cgacatcttg gaccattagc 6661 tccacaggta tcttcttccc tctagtggtc ataacagcag cttcagctac ctctcaattc 6721 aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt 6781 acacacacaa aaaaccaaca cacatccatc ttcgatggat agcgatttta ttatctaact 6841 gctgatcgag tgtagccaga tctagtaatc aattacgggg tcattagttc atagcccata 6901 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 6961 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 7021 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 7081 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 7141 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 7201 catcgctatt accatgctga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 7261 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 7321 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 7381 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat 7441 cagatctttg tcgatcctac catccactcg acacacccgc cagcggccgc //