LOCUS Exported 7756 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3'-to-5' exonuclease. ACCESSION . VERSION . KEYWORDS pGBW-m4134188 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7756) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7756) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7756 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1583..1603 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1607..1672 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 1725..1846 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1736..1755) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1790..1809 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1961..2007 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2037..2173 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2218..2289 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2515..3103) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2595..2614) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3603..4812 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4649..4668 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4816..5415 /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(4816..4835) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5312..5332 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5442..5490 /note="synthetic polyadenylation signal" CDS 5756..6616 /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(5974..5993) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6629..6765 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6910..6981 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6986..7032 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7050..7098 /note="synthetic polyadenylation signal" enhancer 7179..7482 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7484..7686 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7637..7657 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7755..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggaa aatgtaacag gactctttaa agattgtagt aaggtaatca ctgggttaca 61 tcctacacag gcacctacac acctcagtgt tgacactaaa ttcaaaactg aaggtttatg 121 tgttgacata cctggcatac ctaaggacat gacctataga agactcatct ctatgatggg 181 ttttaaaatg aattatcaag ttaatggtta ccctaacatg tttatcaccc gcgaagaagc 241 tataagacat gtacgtgcat ggattggctt cgatgtcgag gggtgtcatg ctactagaga 301 agctgttggt accaatttac ctttacagct aggtttttct acaggtgtta acctagttgc 361 tgtacctaca ggttatgttg atacacctaa taatacagat ttttccagag ttagtgctaa 421 accaccgcct ggagatcaat ttaaacacct cataccactt atgtacaaag gacttccttg 481 gaatgtagtg cgtataaaga ttgtacaaat gttaagtgac acacttaaaa atctctctga 541 cagagtcgta tttgtcttat gggcacatgg ctttgagttg acatctatga agtattttgt 601 gaaaatagga cctgagcgca cctgttgtct atgtgataga cgtgccacat gcttttccac 661 tgcttcagac acttatgcct gttggcatca ttctattgga tttgattacg tctataatcc 721 gtttatgatt gatgttcaac aatggggttt tacaggtaac ctacaaagca accatgatct 781 gtattgtcaa gtccatggta atgcacatgt agctagttgt gatgcaatca tgactaggtg 841 tctagctgtc cacgagtgct ttgttaagcg tgttgactgg actattgaat atcctataat 901 tggtgatgaa ctgaagatta atgcggcttg tagaaaggtt caacacatgg ttgttaaagc 961 tgcattatta gcagacaaat tcccagttct tcacgacatt ggtaacccta aagctattaa 1021 gtgtgtacct caagctgatg tagaatggaa gttctatgat gcacagcctt gtagtgacaa 1081 agcttataaa atagaagaat tattctattc ttatgccaca cattctgaca aattcacaga 1141 tggtgtatgc ctattttgga attgcaatgt cgatagatat cctgctaatt ccattgtttg 1201 tagatttgac actagagtgc tatctaacct taacttgcct ggttgtgatg gtggcagttt 1261 gtatgtaaat aaacatgcat tccacacacc agcttttgat aaaagtgctt ttgttaattt 1321 aaaacaatta ccatttttct attactctga cagtccatgt gagtctcatg gaaaacaagt 1381 agtgtcagat atagattatg taccactaaa gtctgctacg tgtataacac gttgcaattt 1441 aggtggtgct gtctgtagac atcatgctaa tgagtacaga ttgtatctcg atgcttataa 1501 catgatgatc tcagctggct ttagcttgtg ggtttacaaa caatttgata cttataacct 1561 ctggaacact tttacaagac ttgaaaacct ttactttcag tcgatggatt ataaggacca 1621 cgatggagac tacaaagatc atgacattga ttacaaggac gacgatgaca aataagctag 1681 cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat acattgatga 1741 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 1801 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 1861 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa 1921 cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc 1981 tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat 2041 tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga 2101 ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga 2161 aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat 2221 tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt 2281 ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag 2341 ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt 2401 tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg 2461 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2521 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2581 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2641 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2701 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2761 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2821 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2881 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2941 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 3001 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 3061 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 3121 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 3181 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 3241 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 3301 tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc cgaactctcc 3361 aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag gctacctctc 3421 gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct tggcagcgcc 3481 tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg agagaagttc 3541 aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat cggggcgcgc 3601 ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc 3661 tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca 3721 gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta 3781 ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag 3841 gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga 3901 tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg 3961 tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc 4021 tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg 4081 ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc 4141 gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg 4201 aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgagccct 4261 tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc tggggaccct 4321 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 4381 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 4441 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 4501 cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 4561 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 4621 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 4681 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 4741 atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc gtttttggct 4801 tttttgttag acaacatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 4861 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 4921 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 4981 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 5041 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 5101 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggagggcct cctggcgccg 5161 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag 5221 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggccgccga gcgcgccggg 5281 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 5341 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 5401 aagcccggtg cctaggctag tatgtaagcc tagtcttaga taataaaatc gctatccatc 5461 gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag 5521 aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt cagccagtcg 5581 gaatccagct tgggacccag gaagtccaat cgtcagatat tgtactcaag cctggtcacg 5641 gcagcgtacc gatctgttta aacctagata ttgatagtct gatcggtcaa cgtataatcg 5701 agtcctagct tttgcaaaca tctatcaaga gacaggatca gcaggaggct ttcgcatgag 5761 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 5821 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt 5881 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 5941 acgctttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 6001 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 6061 gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 6121 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgattggagg 6181 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 6241 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 6301 agcaatggca acaaccttgc gtaaactatt aactggcgaa ctacttactc tagcttcccg 6361 gcaacagttg atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 6421 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 6481 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 6541 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 6601 gattaagcat tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac 6661 gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact 6721 tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaagatcg tttaaactcg 6781 actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt 6841 tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc 6901 ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta gtggtcataa 6961 cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta gaggccccaa 7021 ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca tccatcttcg 7081 atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta gtaatcaatt 7141 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 7201 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 7261 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 7321 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 7381 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 7441 acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg gttttggcag 7501 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 7561 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 7621 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 7681 agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc cactcgacac 7741 acccgccagc ggccgc //