LOCUS Exported 9437 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression plasmid for SpCas9∆REC3 variant. ACCESSION . VERSION . KEYWORDS pSHS273 - Bacterial expression plasmid for SpCas9∆REC3 variant SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9437) AUTHORS Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA TITLE Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. JOURNAL Nature. 2017 Sep 20. doi: 10.1038/nature24268. PUBMED 28931002 REFERENCE 2 (bases 1 to 9437) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9437 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 376..395 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 479..498 /label=T7 /note="T7 promoter, forward primer" promoter 479..497 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 586..608 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 628..657 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 667..1767 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 1741..1764 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1822..1842 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(5445..5463) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5459..5506 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(5872..5900) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(5927..5945) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6013..6117 /gene="bla" /label=AmpR promoter CDS 6118..6978 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6336..6355) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7149..7737 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7638..7657 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7891..7908 /label=L4440 /note="L4440 vector, forward primer" misc_feature 7923..8062 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7988..8007) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8148..8170 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8164..8355) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc 61 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt 121 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg 181 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt 241 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc 301 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc 361 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc 421 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta 481 atacgactca ctatagggag accacaacgg tttccctcta gtgccggctc cggagagctc 541 tttaattaag cggccgccct gcaggactcg agttctagaa ataattttgt ttaactttaa 601 gaaggagata tacatatgaa atcttctcac catcaccatc accatcacca tcaccatggt 661 tcttctatga aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat 721 aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt 781 gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct 841 gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct 901 gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta 961 cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat 1021 aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa 1081 gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc 1141 tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac 1201 attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac 1261 ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt 1321 aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc 1381 agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg 1441 ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa 1501 gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa 1561 ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt 1621 gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc 1681 gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc 1741 gatgaagccc tgaaagacgc gcagactaat tcgagctcga acaacaacaa caataacaat 1801 aacaacaacc tcgggatcga ggaaaacctg tacttccaat ccaatgcaat ggataagaaa 1861 tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa 1921 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa 1981 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc 2041 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag 2101 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct 2161 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat 2221 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat 2281 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt 2341 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta 2401 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt 2461 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat 2521 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg 2581 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta 2641 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat 2701 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat 2761 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc 2821 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca 2881 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat 2941 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat 3001 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc 3061 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga 3121 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg 3181 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg 3241 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa 3301 ggtgcttcag ctcaatcatt tattgaacgc atgacaaacg gcggtagtgt gtctggacaa 3361 ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt 3421 attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca 3481 gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat 3541 tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt 3601 aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc 3661 caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat 3721 gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc 3781 ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc 3841 aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag 3901 tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt 3961 atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat 4021 agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt 4081 accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt 4141 gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct 4201 ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat 4261 gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat 4321 ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag 4381 attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa 4441 gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag 4501 aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg 4561 gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt 4621 ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag 4681 ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa 4741 aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt 4801 aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt 4861 gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta 4921 tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa 4981 ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt 5041 tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa 5101 catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg 5161 aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat 5221 acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat 5281 gaaacacgca ttgatttgag tcagctagga ggtgactaat aacattggaa gtggataacg 5341 gatccgcgat cgcggcgcgc cacctggtgg ccggccggta ccacgcgtgc gcgctgatcc 5401 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact 5461 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac 5521 tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc 5581 caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt gctccgagaa 5641 cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca tagtgactgg 5701 cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc ataaccaagc 5761 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt 5821 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct 5881 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc 5941 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 6001 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 6061 ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag gaagagtatg 6121 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 6181 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 6241 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 6301 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 6361 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 6421 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 6481 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 6541 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 6601 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 6661 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 6721 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 6781 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 6841 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 6901 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 6961 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 7021 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 7081 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 7141 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 7201 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 7261 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 7321 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 7381 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 7441 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 7501 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 7561 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 7621 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 7681 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 7741 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 7801 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 7861 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 7921 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca 7981 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct 8041 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg 8101 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 8161 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc 8221 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag 8281 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt 8341 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat 8401 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt 8461 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat 8521 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca 8581 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc 8641 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt 8701 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt 8761 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg 8821 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc 8881 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt 8941 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc 9001 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg 9061 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg 9121 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc 9181 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg 9241 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac 9301 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg 9361 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg 9421 aataccgcaa gcgacag //