LOCUS Exported 8693 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3'-to-5' exonuclease. ACCESSION . VERSION . KEYWORDS pGBW-m4133523 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8693) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8693) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8693 /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 1643..1720 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" CDS 1748..1768 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 2233..2354 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2244..2263) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2298..2317 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2469..2515 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2545..2681 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2726..2797 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3023..3611) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3103..3122) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4111..5320 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5157..5176 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 5324..6352 /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 6379..6427 /note="synthetic polyadenylation signal" CDS 6693..7553 /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(6911..6930) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 7566..7702 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7847..7918 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7923..7969 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7987..8035 /note="synthetic polyadenylation signal" enhancer 8116..8419 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 8421..8623 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 8574..8594 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 8692..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 gacggcgaat 1621 cccggggctg ataaatccct ggaaaaggag atggaagaag aactttattg cagtgtctgc 1681 ggctaaccgg ttcaaaaaga tcagttcaag cggcgcccta gattacgaca ttcctacgac 1741 cgcctccgag aatctctatt tccagggtga atttggcttg gcacaacatg acgaggccgt 1801 cgataacaag tttaataaag aacagcagaa cgctttctat gagatccttc acttaccaaa 1861 tctgaacgaa gagcaacgca acgctttcat tcagagcctg aaagacgatc cctcgcagtc 1921 tgcaaatctc ctggccgaag ccaagaagct gaatgacgca caggctccaa aagttgataa 1981 caagtttaac aaggagcagc agaatgcttt ctacgagata ttgcaccttc ctaatctgaa 2041 tgaggaacaa agaaatgcat ttatccagtc cctaaaagac gatccatcac agagtgccaa 2101 cctgcttgcg gaagccaaaa agctcaatga tgcccaggcc ccgaaagtag acgcgaacca 2161 tcaataagga tccgcaggcc tctgctagct tgactgactg agatacagcg taccttcagc 2221 tcacagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 2281 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 2341 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 2401 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtattggc ccatctctat 2461 cggtatcgta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc 2521 ctcgggccgg attgctatct accggcattg gcgcagaaaa aaatgcctga tgcgacgctg 2581 cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc 2641 cacttccata cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag ctatcctgca 2701 ggcgatctct cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg 2761 ggtcagaagt agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta 2821 tcgaaactta attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg 2881 actacgctca gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc 2941 cgattacgag tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc aggaaccgta 3001 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 3061 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 3121 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 3181 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 3241 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 3301 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 3361 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 3421 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 3481 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 3541 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 3601 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 3661 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 3721 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 3781 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 3841 tagttgcatt taaatttccg aactctccaa ggccctcgtc ggaaaatctt caaacctttc 3901 gtccgatcca tcttgcaggc tacctctcga acgaactatc gcaagtctct tggccggcct 3961 tgcgccttgg ctattgcttg gcagcgccta tcgccaggta ttactccaat cccgaatatc 4021 cgagatcggg atcacccgag agaagttcaa cctacatcct caatcccgat ctatccgaga 4081 tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg ccgggttttg gcgcctcccg 4141 cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga cgaagggcgc aggagcgtcc 4201 tgatccttcc gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga 4261 accccagtat cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt 4321 tttctttcca gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga 4381 gggatctccg tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca 4441 gctagttccg tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg 4501 tcacttggtg agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc 4561 ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg 4621 ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga 4681 atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg 4741 gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg gaaagctctt attcgggtga 4801 gatgggctgg gcaccatctg gggaccctga cgtgaagttt gtcactgact ggagaactcg 4861 gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc gttgggcagt gcacccgtac 4921 ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc 4981 agggtggggc cacctgccgg taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg 5041 ttcgggccta gggtaggctc tcctgaatcg acaggcgccg gacctctggt gaggggaggg 5101 ataagtgagg cgtcagtttc tttggtcggt tttatgtacc tatcttctta agtagctgaa 5161 gctccggttt tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc 5221 accttttgaa atgtaatcat ttgggtcaat atgtaatttt cagtgttaga cttgtaaatt 5281 gtccgctaaa ttctggccgt ttttggcttt tttgttagac aacatgggta aaaagcctga 5341 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct 5401 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg 5461 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg 5521 gcactttgca tccgccgcgc tcccgattcc ggaagtgctt gacattgggg agttcagcga 5581 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga 5641 aaccgaactg cccgctgttc tgcagccggt cgcggaggca atggatgcca tcgctgccgc 5701 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac 5761 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt 5821 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc 5881 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct 5941 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc 6001 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca 6061 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccccggc tccgggcgta 6121 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga 6181 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg 6241 gcgtacacaa atcgcccgca gaagcgccgc cgtctggacc gatggctgtg tagaagtact 6301 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aagctagtat 6361 gtaagcctag tcttagataa taaaatcgct atccatcgaa gatggatgtg tgttggtttt 6421 ttgtgtgtgt aacgctaggc gcgcctggtg taccgagaac gatcctctca gtgcgagtct 6481 cgacgatcca tatcgttgct tggcagtcag ccagtcggaa tccagcttgg gacccaggaa 6541 gtccaatcgt cagatattgt actcaagcct ggtcacggca gcgtaccgat ctgtttaaac 6601 ctagatattg atagtctgat cggtcaacgt ataatcgagt cctagctttt gcaaacatct 6661 atcaagagac aggatcagca ggaggctttc gcatgagtat tcaacatttc cgtgtcgccc 6721 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6781 aagtaaaaga tgctgaagat cagttgggtg cgcgagtggg ttacatcgaa ctggatctca 6841 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ctttccaatg atgagcactt 6901 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6961 gtcgccgcat acactattct cagaatgact tggttgagta ttcaccagtc acagaaaagc 7021 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 7081 acactgcggc caacttactt ctgacaacga ttggaggacc gaaggagcta accgcttttt 7141 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 7201 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca accttgcgta 7261 aactattaac tggcgaacta cttactctag cttcccggca acagttgata gactggatgg 7321 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 7381 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 7441 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 7501 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taaccgattc 7561 taggtgcatt ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta tactcccaca 7621 tatgccagat tcagcaacgg atacggcttc cccaacttgc ccacttccat acgtgtcctc 7681 cttaccagaa atttatcctt aagatcgttt aaactcgact ctggctctat cgaatctccg 7741 tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg 7801 tcagctatcg tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt 7861 agctccacag gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa 7921 ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc 7981 gttacacaca caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta 8041 actgctgatc gagtgtagcc agatctagta atcaattacg gggtcattag ttcatagccc 8101 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 8161 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 8221 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 8281 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 8341 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 8401 agtcatcgct attaccatgc tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 8461 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 8521 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 8581 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca 8641 gatcagatct ttgtcgatcc taccatccac tcgacacacc cgccagcggc cgc //