LOCUS Exported 7711 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 3'-to-5' exonuclease. ACCESSION . VERSION . KEYWORDS pGBW-m4134054 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7711) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7711) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7711 /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..1627 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1680..1801 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1691..1710) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1745..1764 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1916..1962 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1992..2128 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2173..2244 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2470..3058) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2550..2569) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3558..4767 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4604..4623 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4771..5370 /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(4771..4790) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5267..5287 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5397..5445 /note="synthetic polyadenylation signal" CDS 5711..6571 /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(5929..5948) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6584..6720 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6865..6936 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6941..6987 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7005..7053 /note="synthetic polyadenylation signal" enhancer 7134..7437 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7439..7641 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7592..7612 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7710..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 tttcagtcgc accatcacca 1621 tcaccattaa gctagcttga ctgactgaga tacagcgtac cttcagctca cagacatgat 1681 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 1741 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 1801 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 1861 ttaaagcaag taaaacctct acaaatgtgg tattggccca tctctatcgg tatcgtagca 1921 taaccccttg gggcctctaa acgggtcttg aggggttttt tgtgcccctc gggccggatt 1981 gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact 2041 cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt 2101 gtcctcctta ccagaaattt atccttaagg tcgtcagcta tcctgcaggc gatctctcga 2161 tttcgatcaa gacattcctt taatggtctt ttctggacac cactaggggt cagaagtagt 2221 tcatcaaact ttcttccctc cctaatctca ttggttacct tgggctatcg aaacttaatt 2281 aaccagtcaa gtcagctact tggcgagatc gacttgtctg ggtttcgact acgctcagaa 2341 ttgcgtcagt caagttcgat ctggtccttg ctattgcacc cgttctccga ttacgagttt 2401 catttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 2461 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 2521 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 2581 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 2641 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 2701 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 2761 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 2821 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 2881 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 2941 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 3001 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 3061 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 3121 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 3181 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 3241 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcatttaa 3301 atttccgaac tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc cgatccatct 3361 tgcaggctac ctctcgaacg aactatcgca agtctcttgg ccggccttgc gccttggcta 3421 ttgcttggca gcgcctatcg ccaggtatta ctccaatccc gaatatccga gatcgggatc 3481 acccgagaga agttcaacct acatcctcaa tcccgatcta tccgagatcc gaggaatatc 3541 gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct 3601 cgtcacggcg agcgctgcca cgtcagacga agggcgcagg agcgtcctga tccttccgcc 3661 cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag 3721 cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag 3781 agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg 3841 ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg 3901 cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt 3961 agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag 4021 cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg 4081 ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt 4141 gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc 4201 aaggtcttga gcccttcgct aatgcgggaa agctcttatt cgggtgagat gggctgggca 4261 ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt 4321 tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg 4381 cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg gtggggccac 4441 ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc gggcctaggg 4501 taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata agtgaggcgt 4561 cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct ccggttttga 4621 actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg 4681 taatcatttg ggtcaatatg taattttcag tgttagactt gtaaattgtc cgctaaattc 4741 tggccgtttt tggctttttt gttagacaac atgaccgagt acaagcccac ggtgcgcctc 4801 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 4861 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 4921 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 4981 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 5041 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggag 5101 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc 5161 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcc 5221 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 5281 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 5341 tggtgcatga cccgcaagcc cggtgcctag gctagtatgt aagcctagtc ttagataata 5401 aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc 5461 gcctggtgta ccgagaacga tcctctcagt gcgagtctcg acgatccata tcgttgcttg 5521 gcagtcagcc agtcggaatc cagcttggga cccaggaagt ccaatcgtca gatattgtac 5581 tcaagcctgg tcacggcagc gtaccgatct gtttaaacct agatattgat agtctgatcg 5641 gtcaacgtat aatcgagtcc tagcttttgc aaacatctat caagagacag gatcagcagg 5701 aggctttcgc atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 5761 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 5821 gttgggtgcg cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 5881 ttttcgcccc gaagaacgct ttccaatgat gagcactttt aaagttctgc tatgtggcgc 5941 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 6001 gaatgacttg gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt 6061 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 6121 gacaacgatt ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 6181 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 6241 caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg gcgaactact 6301 tactctagct tcccggcaac agttgataga ctggatggag gcggataaag ttgcaggacc 6361 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 6421 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 6481 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 6541 gataggtgcc tcactgatta agcattggta accgattcta ggtgcattgg cgcagaaaaa 6601 aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc agcaacggat 6661 acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat ttatccttaa 6721 gatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct tacgcgaaca 6781 gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc agcttacctt 6841 tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt atcttcttcc 6901 ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc 6961 gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca aaaaaccaac 7021 acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga gtgtagccag 7081 atctagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca 7141 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 7201 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 7261 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 7321 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 7381 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatgctg 7441 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca 7501 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 7561 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg 7621 gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt gtcgatccta 7681 ccatccactc gacacacccg ccagcggccg c //