LOCUS Exported 8187 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3'-to-5' exonuclease. ACCESSION . VERSION . KEYWORDS pGBW-m4134022 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8187) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8187) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8187 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1589..1609 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1610..1651 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" polyA_signal 1720..1841 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1731..1750) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1785..1804 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1956..2002 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2032..2168 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2213..2284 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2510..3098) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2590..2609) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3598..4807 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4644..4663 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4811..5839 /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 5866..5914 /note="synthetic polyadenylation signal" CDS 6180..7040 /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(6398..6417) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 7053..7189 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7341..7412 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7417..7463 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7481..7529 /note="synthetic polyadenylation signal" enhancer 7610..7913 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7915..8117 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 8068..8088 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 8186..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggcc gaaaatgtca ctggcttatt taaggattgc agtaaagtga tcacagggtt 61 gcacccgacc caagcgccta cgcatctttc agtagacacc aagttcaaaa ctgagggact 121 ctgtgtggac ataccaggta ttcccaagga tatgacatac agacgcctga tctcgatgat 181 gggcttcaag atgaactatc aggttaacgg ttaccccaat atgtttatta cccgagagga 241 agctatccgg cacgtgcgtg cctggatagg gtttgacgtc gagggctgtc atgcaactag 301 ggaagctgta ggaacaaatc tgccactgca actcggcttc tctaccggag tcaacctagt 361 tgccgtgcct accgggtatg tggatacacc aaacaatact gattttagca gagtctccgc 421 aaaaccccct cccggggacc agttcaaaca cttgatcccc ctgatgtaca agggtttacc 481 atggaacgtg gtgcggatta aaatagttca gatgctcagc gacacactga agaatttaag 541 tgatagagtt gtgtttgtac tttgggccca tggcttcgag ctgacctcta tgaagtattt 601 cgttaaaatt ggacccgaac gaacgtgctg cctgtgtgac cggagggcga cgtgtttctc 661 aactgcttcc gatacctatg cctgctggca ccatagcatc ggctttgatt acgtgtacaa 721 cccttttatg attgacgtgc agcagtgggg attcactggg aacctacagt cgaatcacga 781 tctttactgt caggtccatg ggaacgcaca cgttgctagc tgcgacgcaa tcatgacacg 841 ttgccttgct gtccacgagt gttttgtgaa acgagtggac tggactattg agtatcctat 901 cattggcgac gaactgaaga taaatgcggc ttgtaggaag gtgcagcata tggtcgtcaa 961 agccgccctc ttagctgata agttcccggt cctgcacgat attggcaatc caaaggccat 1021 caaatgcgta ccccaggcag atgttgagtg gaagttttac gacgcgcaac catgctccga 1081 taaggcctat aaaatcgaag agctcttcta cagttatgcc acccattctg acaagtttac 1141 cgacggtgtg tgtttgtttt ggaattgcaa cgtggatcgc taccctgcaa actctatcgt 1201 ttgtcgcttc gacacacgcg tattgtccaa tctgaatcta cccggatgcg acggaggttc 1261 actgtatgtg aacaagcacg ccttccacac ccccgcattc gataaatccg cctttgtcaa 1321 ccttaaacaa ttacctttct tttactacag cgactctcct tgcgaatccc atggaaagca 1381 agtagtgagt gatatcgatt acgtcccact caaaagtgca acatgtatta cgagatgcaa 1441 tctcggtggt gcagtctgta gacaccatgc caatgaatat cggctgtatt tggacgccta 1501 taacatgatg atttcagctg gcttctctct ttgggtttac aagcagtttg acacgtataa 1561 tctgtggaac acctttacaa ggctgcaaga aaacctttac tttcagtcgg ggaaacctat 1621 acccaatcca cttctcggct tagattctac ctaaggatcc gcaggcctct gctagcttga 1681 ctgactgaga tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg 1741 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 1801 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 1861 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 1921 acaaatgtgg tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa 1981 acgggtcttg aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg 2041 cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag 2101 caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt 2161 atccttaagg tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt 2221 taatggtctt ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc 2281 cctaatctca ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact 2341 tggcgagatc gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat 2401 ctggtccttg ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa 2461 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 2521 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2581 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 2641 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 2701 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 2761 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 2821 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 2881 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 2941 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3001 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3061 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3121 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3181 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3241 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3301 cagcgatctg tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc 3361 cctcgtcgga aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg 3421 aactatcgca agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg 3481 ccaggtatta ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct 3541 acatcctcaa tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc 3601 ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca 3661 cgtcagacga agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc 3721 ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg 3781 ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa 3841 gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt 3901 atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg 3961 gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc 4021 ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag 4081 ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc 4141 aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc 4201 gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct 4261 aatgcgggaa agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt 4321 gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg 4381 cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac 4441 gtcacccgtt ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta 4501 ggcttttctc cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca 4561 ggcgccggac ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt 4621 atgtacctat cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc 4681 gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg 4741 taattttcag tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt 4801 gttagacaac atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 4861 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4921 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4981 tttctacaaa gatcgttatg tttatcggca ctttgcatcc gccgcgctcc cgattccgga 5041 agtgcttgac attggggagt tcagcgagag cctgacctat tgcatctccc gccgtgcaca 5101 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 5161 ggaggcaatg gatgccatcg ctgccgccga tcttagccag acgagcgggt tcggcccatt 5221 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 5281 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 5341 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 5401 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 5461 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 5521 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 5581 tgcaggatcg ccccggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 5641 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 5701 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgccgccgt 5761 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 5821 tccgagggca aaggaataag ctagtatgta agcctagtct tagataataa aatcgctatc 5881 catcgaagat ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac 5941 cgagaacgat cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca 6001 gtcggaatcc agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt 6061 cacggcagcg taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata 6121 atcgagtcct agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca 6181 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 6241 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc 6301 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 6361 aagaacgctt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 6421 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 6481 ttgagtattc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6541 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg 6601 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 6661 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 6721 ctgtagcaat ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt 6781 cccggcaaca gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 6841 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 6901 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 6961 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 7021 cactgattaa gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg 7081 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc 7141 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag atcccgaatc 7201 gtttaaactc gactctggct ctatcgaatc tccgtcgttt cgagcttacg cgaacagccg 7261 tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct atcgtcagct tacctttttg 7321 gcagcgatcg cggctcccga catcttggac cattagctcc acaggtatct tcttccctct 7381 agtggtcata acagcagctt cagctacctc tcaattcaaa aaacccctca agacccgttt 7441 agaggcccca aggggttatg ctatcaatcg ttgcgttaca cacacaaaaa accaacacac 7501 atccatcttc gatggatagc gattttatta tctaactgct gatcgagtgt agccagatct 7561 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 7621 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 7681 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 7741 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 7801 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 7861 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atgctgatgc 7921 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 7981 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 8041 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 8101 tctatataag cagagctggt ttagtgaacc gtcagatcag atctttgtcg atcctaccat 8161 ccactcgaca cacccgccag cggccgc //