LOCUS Exported 7732 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3'-to-5' exonuclease. ACCESSION . VERSION . KEYWORDS pGBW-m4134433 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7732) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7732) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7732 /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 1604..1648 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" polyA_signal 1701..1822 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1712..1731) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1766..1785 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1937..1983 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2013..2149 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2194..2265 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2491..3079) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2571..2590) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3579..4788 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4625..4644 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4792..5391 /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(4792..4811) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5288..5308 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5418..5466 /note="synthetic polyadenylation signal" CDS 5732..6592 /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(5950..5969) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6605..6741 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6886..6957 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6962..7008 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7026..7074 /note="synthetic polyadenylation signal" enhancer 7155..7458 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7460..7662 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7613..7633 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7731..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 tcgaaagaaa ccgcggcagc 1621 taagttcgag aggcagcaca tggacagtta agctagcttg actgactgag atacagcgta 1681 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat 1741 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 1801 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 1861 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc 1921 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 1981 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg 2041 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc 2101 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct 2161 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca 2221 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc 2281 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct 2341 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac 2401 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag 2461 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 2521 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 2581 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 2641 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 2701 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 2761 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 2821 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 2881 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 2941 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3001 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3061 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 3181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 3241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 3301 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca 3361 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg 3421 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc 3481 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct 3541 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc 3601 gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag 3661 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg 3721 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg 3781 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat 3841 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt 3901 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc 3961 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg 4021 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc 4081 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg 4141 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg 4201 catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga aagctcttat 4261 tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt cactgactgg 4321 agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc 4381 acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct 4441 tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg 4501 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga 4561 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag 4621 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt 4681 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact 4741 tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa catgaccgag 4801 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc 4861 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc 4921 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag 4981 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa 5041 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg 5101 gccgcgcagc aacagatgga gggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg 5161 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc 5221 gtgctccccg gagtggaggc cgccgagcgc gccggggtgc ccgccttcct ggagacctcc 5281 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag 5341 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta ggctagtatg 5401 taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt 5461 tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc 5521 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag 5581 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc 5641 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta 5701 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct 5761 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 5821 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa 5881 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt 5941 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 6001 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca 6061 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6121 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt 6181 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 6241 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa 6301 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga 6361 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 6421 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 6481 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 6541 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct 6601 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat 6661 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc 6721 ttaccagaaa tttatcctta agatcgttta aactcgactc tggctctatc gaatctccgt 6781 cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt 6841 cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta 6901 gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat 6961 tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg 7021 ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattatctaa 7081 ctgctgatcg agtgtagcca gatctagtaa tcaattacgg ggtcattagt tcatagccca 7141 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 7201 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 7261 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 7321 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 7381 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 7441 gtcatcgcta ttaccatgct gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 7501 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 7561 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 7621 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag 7681 atcagatctt tgtcgatcct accatccact cgacacaccc gccagcggcc gc //