LOCUS Exported 7168 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 endoRNAse. ACCESSION . VERSION . KEYWORDS pGBW-m4133322 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7168) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7168) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7168 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1046..1066 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1067..1084 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1137..1258 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1148..1167) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1202..1221 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1373..1419 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1449..1585 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1630..1701 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1927..2515) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2007..2026) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3015..4224 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4061..4080 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4228..4827 /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(4228..4247) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4724..4744 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4854..4902 /note="synthetic polyadenylation signal" CDS 5168..6028 /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(5386..5405) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6041..6177 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6322..6393 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6398..6444 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6462..6510 /note="synthetic polyadenylation signal" enhancer 6591..6894 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6896..7098 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7049..7069 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgtca ctggagaacg ttgctttcaa tgtagtgaac aaggggcact ttgacggaca 61 gcaaggcgaa gtgccggtca gtatcataaa taatacggtg tacaccaaag tcgatggtgt 121 tgacgtggag ttatttgaaa acaagacaac tttgcctgtc aatgtggcct tcgagctgtg 181 ggcaaaacgc aacattaagc cagtacccga agtgaaaatc ctcaacaatc ttggcgttga 241 tattgccgca aacactgtta tctgggatta taagcgagat gcgcctgccc atatatccac 301 cattggagtg tgttctatga cagacatcgc taaaaagccc accgagacaa tttgcgcgcc 361 actcacggta ttctttgacg ggagggtcga cggtcaggtg gatctgttta gaaatgcccg 421 taacggcgta cttattactg aaggaagcgt gaaaggccta cagcccagtg ttgggccgaa 481 gcaagcaagc ctaaatggag tcacactgat cggagaggct gtgaaaaccc agttcaacta 541 ctataagaag gtcgacggcg tggttcagca gctgcctgag acatacttta cccaatcgcg 601 gaatctgcaa gaatttaagc ccagatccca gatggagatt gacttcttgg aactggccat 661 ggatgagttc atcgaacggt ataagctcga gggctacgcc ttcgagcaca tagtttatgg 721 ggatttttct cattcacagt taggcggtct gcacttgctc atcggattag cgaaacgctt 781 taaagagagc ccattcgaac ttgaagactt tatcccaatg gactcaactg tgaagaatta 841 cttcataact gacgctcaga cagggtccag caagtgcgtc tgttctgtta ttgatttgct 901 acttgatgat ttcgtggaga ttattaaatc acaggacctg tccgtggttt cgaaggtcgt 961 gaaagtaacc atcgactata cggaaattag tttcatgctt tggtgtaagg atggccatgt 1021 ggagacattt tatcctaagc tccaggaaaa cctttacttt cagtcgcacc atcaccatca 1081 ccattaagct agcttgactg actgagatac agcgtacctt cagctcacag acatgataag 1141 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg 1201 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa 1261 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta 1321 aagcaagtaa aacctctaca aatgtggtat tggcccatct ctatcggtat cgtagcataa 1381 ccccttgggg cctctaaacg ggtcttgagg ggttttttgt gcccctcggg ccggattgct 1441 atctaccggc attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc 1501 acatatgcca gattcagcaa cggatacggc ttccccaact tgcccacttc catacgtgtc 1561 ctccttacca gaaatttatc cttaaggtcg tcagctatcc tgcaggcgat ctctcgattt 1621 cgatcaagac attcctttaa tggtcttttc tggacaccac taggggtcag aagtagttca 1681 tcaaactttc ttccctccct aatctcattg gttaccttgg gctatcgaaa cttaattaac 1741 cagtcaagtc agctacttgg cgagatcgac ttgtctgggt ttcgactacg ctcagaattg 1801 cgtcagtcaa gttcgatctg gtccttgcta ttgcacccgt tctccgatta cgagtttcat 1861 ttaaatcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 1921 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 1981 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 2041 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 2101 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 2161 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 2221 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 2281 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 2341 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 2401 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2461 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 2521 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 2581 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 2641 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 2701 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg catttaaatt 2761 tccgaactct ccaaggccct cgtcggaaaa tcttcaaacc tttcgtccga tccatcttgc 2821 aggctacctc tcgaacgaac tatcgcaagt ctcttggccg gccttgcgcc ttggctattg 2881 cttggcagcg cctatcgcca ggtattactc caatcccgaa tatccgagat cgggatcacc 2941 cgagagaagt tcaacctaca tcctcaatcc cgatctatcc gagatccgag gaatatcgaa 3001 atcggggcgc gcctggcctc cgcgccgggt tttggcgcct cccgcgggcg cccccctcgt 3061 cacggcgagc gctgccacgt cagacgaagg gcgcaggagc gtcctgatcc ttccgcccgg 3121 acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag 3181 aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc 3241 ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc 3301 ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag 3361 ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt ggtgagtagc 3421 gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt 3481 gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg 3541 gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa gacgcttgtg 3601 aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca agaacccaag 3661 gtcttgagcc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg ctgggcacca 3721 tctggggacc ctgacgtgaa gtttgtcact gactggagaa ctcggtttgt cgtctgttgc 3781 gggggcggca gttatggcgg tgccgttggg cagtgcaccc gtacctttgg gagcgcgcgc 3841 cctcgtcgtg tcgtgacgtc acccgttctg ttggcttata atgcagggtg gggccacctg 3901 ccggtaggtg tgcggtaggc ttttctccgt cgcaggacgc agggttcggg cctagggtag 3961 gctctcctga atcgacaggc gccggacctc tggtgagggg agggataagt gaggcgtcag 4021 tttctttggt cggttttatg tacctatctt cttaagtagc tgaagctccg gttttgaact 4081 atgcgctcgg ggttggcgag tgtgttttgt gaagtttttt aggcaccttt tgaaatgtaa 4141 tcatttgggt caatatgtaa ttttcagtgt tagacttgta aattgtccgc taaattctgg 4201 ccgtttttgg cttttttgtt agacaacatg accgagtaca agcccacggt gcgcctcgcc 4261 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 4321 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 4381 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 4441 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 4501 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggagggc 4561 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 4621 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggccgcc 4681 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac 4741 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 4801 tgcatgaccc gcaagcccgg tgcctaggct agtatgtaag cctagtctta gataataaaa 4861 tcgctatcca tcgaagatgg atgtgtgttg gttttttgtg tgtgtaacgc taggcgcgcc 4921 tggtgtaccg agaacgatcc tctcagtgcg agtctcgacg atccatatcg ttgcttggca 4981 gtcagccagt cggaatccag cttgggaccc aggaagtcca atcgtcagat attgtactca 5041 agcctggtca cggcagcgta ccgatctgtt taaacctaga tattgatagt ctgatcggtc 5101 aacgtataat cgagtcctag cttttgcaaa catctatcaa gagacaggat cagcaggagg 5161 ctttcgcatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 5221 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 5281 gggtgcgcga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 5341 tcgccccgaa gaacgctttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 5401 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 5461 tgacttggtt gagtattcac cagtcacaga aaagcatctt acggatggca tgacagtaag 5521 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 5581 aacgattgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 5641 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 5701 cacgatgcct gtagcaatgg caacaacctt gcgtaaacta ttaactggcg aactacttac 5761 tctagcttcc cggcaacagt tgatagactg gatggaggcg gataaagttg caggaccact 5821 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 5881 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 5941 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 6001 aggtgcctca ctgattaagc attggtaacc gattctaggt gcattggcgc agaaaaaaat 6061 gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc aacggatacg 6121 gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta tccttaagat 6181 cgtttaaact cgactctggc tctatcgaat ctccgtcgtt tcgagcttac gcgaacagcc 6241 gtggcgctca tttgctcgtc gggcatcgaa tctcgtcagc tatcgtcagc ttaccttttt 6301 ggcagcgatc gcggctcccg acatcttgga ccattagctc cacaggtatc ttcttccctc 6361 tagtggtcat aacagcagct tcagctacct ctcaattcaa aaaacccctc aagacccgtt 6421 tagaggcccc aaggggttat gctatcaatc gttgcgttac acacacaaaa aaccaacaca 6481 catccatctt cgatggatag cgattttatt atctaactgc tgatcgagtg tagccagatc 6541 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 6601 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 6661 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 6721 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 6781 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 6841 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catgctgatg 6901 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 6961 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 7021 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 7081 gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc gatcctacca 7141 tccactcgac acacccgcca gcggccgc //