LOCUS HBT-pcoCas9. 7940 bp DNA circular SYN 01-JAN-1980 DEFINITION Plasmid for transient expression of plant codon-optimized Cas9 in plant cells. Use together with pUC119-gRNA. ACCESSION . VERSION . KEYWORDS HBT-pcoCas9. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7940) AUTHORS Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J. TITLE Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. JOURNAL Nat. Biotechnol. 2013;31:688-91. PUBMED 23929339 REFERENCE 2 (bases 1 to 7940) AUTHORS Sheen Lab / Addgene #52254 TITLE Direct Submission REFERENCE 3 (bases 1 to 7940) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat. Biotechnol."; date: "2013"; volume: "31"; pages: "688-91" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..7940 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind complement(39..55) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(63..79) /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(87..117) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(132..153) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(441..1029) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1203..2060) /label=AmpR /note="beta-lactamase" promoter complement(2061..2165) /label=AmpR promoter primer_bind 2639..2655 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 2722..3231 /label=35SPPDK promoter /note="hybrid promoter consisting of the cauliflower mosaic virus 35S enhancer fused to the maize C4PPDK basal promoter (Yoo et al., 2007)" CDS 3240..3242 /codon_start=1 /product="start codon" /label=start codon /note="ATG" /translation="M" CDS 3243..3290 /label=2xFLAG /note="two tandem FLAG(R) epitope tags" CDS 3297..3317 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" intron 4779..4967 /label=IV2 intron /note="modified second intron of the potato ST-LS1 gene (Vancanneyt et al., 1990)" CDS 5133..7631 /label=Cas9(C) /note="C-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" CDS 7632..7679 /label=nucleoplasmin NLS /note="bipartite nuclear localization signal from nucleoplasmin" terminator 7688..7940 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" ORIGIN 1 cgatgataag ctgtcaaaca tgagaattcg taatcatggt catagctgtt tcctgtgtga 61 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 121 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 181 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 241 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 301 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 361 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 421 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 481 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 541 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 601 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 661 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 721 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 781 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 841 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 901 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 961 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 1021 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 1081 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 1141 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 1201 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 1261 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 1321 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 1381 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 1441 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 1501 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 1561 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 1621 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 1681 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 1741 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 1801 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 1861 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 1921 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 1981 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 2041 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 2101 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 2161 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 2221 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac 2281 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat 2341 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg 2401 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata 2461 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc 2521 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 2581 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 2641 aaaacgacgg ccagtgccaa gctctcgaga agcttactcc aagaatatca aagatacagt 2701 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct 2761 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg 2821 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga 2881 cagtggtccc aaagatggac ccccacccac aaggagcatc gtggaaaaag aagacgttcc 2941 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc 3001 acaatcccac tatccttcgc cccaagcttg ggcccaagct tgggtcgcgc cccacggatg 3061 gtataagaat aaaggcattc cgcgtgcagg attcacccgt tcgcctctca ccttttcgct 3121 gtactctctc gccacacaca ccccctctcc agctccgttg gagctccgga cagcagcagg 3181 cgcggggcgg tcacgtagta agcagctctc ggctccctct ccccttgctc cgtggatcca 3241 tggattacaa ggatgatgat gataaggatt acaaggatga tgatgataag atggctccaa 3301 agaagaagag aaaggttgga atccacggag ttccagctgc tgataagaag tactctatcg 3361 gacttgacat cggaaccaac tctgttggat gggctgttat caccgatgag tacaaggttc 3421 catctaagaa gttcaaggtt cttggaaaca ccgatagaca ctctatcaag aagaacctta 3481 tcggtgctct tcttttcgat tctggagaga ccgctgaggc taccagattg aagagaaccg 3541 ctagaagaag atacaccaga agaaagaaca gaatctgcta ccttcaggaa atcttctcta 3601 acgagatggc taaggttgat gattctttct tccacagact tgaggagtct ttccttgttg 3661 aggaggataa gaagcacgag agacacccaa tcttcggaaa catcgttgat gaggttgctt 3721 accacgagaa gtacccaacc atctaccacc ttagaaagaa gttggttgat tctaccgata 3781 aggctgatct tagacttatc taccttgctc ttgctcacat gatcaagttc agaggacact 3841 tccttatcga gggagacctt aacccagata actctgatgt tgataagttg ttcatccagc 3901 ttgttcagac ctacaaccag cttttcgagg agaacccaat caacgcttct ggagttgatg 3961 ctaaggctat cctttctgct agactttcta agtctcgtag acttgagaac cttatcgctc 4021 agcttccagg agagaagaag aacggacttt tcggaaacct tatcgctctt tctcttggac 4081 ttaccccaaa cttcaagtct aacttcgatc ttgctgagga tgctaagttg cagctttcta 4141 aggataccta cgatgatgat cttgataacc ttcttgctca gatcggagat cagtacgctg 4201 atcttttcct tgctgctaag aacctttctg atgctatcct tctttctgac atccttagag 4261 ttaacaccga gatcaccaag gctccacttt ctgcttctat gatcaagaga tacgatgagc 4321 accaccagga tcttaccctt ttgaaggctc ttgttagaca gcagcttcca gagaagtaca 4381 aggaaatctt cttcgatcag tctaagaacg gatacgctgg atacatcgat ggaggagctt 4441 ctcaggagga gttctacaag ttcatcaagc caatccttga gaagatggat ggaaccgagg 4501 agcttcttgt taagttgaac agagaggatc ttcttagaaa gcagagaacc ttcgataacg 4561 gatctatccc acaccagatc caccttggag agcttcacgc tatccttcgt agacaggagg 4621 atttctaccc attcttgaag gataacagag agaagatcga gaagatcctt accttcagaa 4681 tcccatacta cgttggacca cttgctagag gaaactctcg tttcgcttgg atgaccagaa 4741 agtctgagga gaccatcacc ccttggaact tcgaggaggt aagtttctgc ttctaccttt 4801 gatatatata taataattat cattaattag tagtaatata atatttcaaa tatttttttc 4861 aaaataaaag aatgtagtat atagcaattg cttttctgta gtttataagt gtgtatattt 4921 taatttataa cttttctaat atatgaccaa aatttgttga tgtgcaggtt gttgataagg 4981 gagcttctgc tcagtctttc atcgagagaa tgaccaactt cgataagaac cttccaaacg 5041 agaaggttct tccaaagcac tctcttcttt acgagtactt caccgtttac aacgagctta 5101 ccaaggttaa gtacgttacc gagggaatga gaaagccagc tttcctttct ggagagcaga 5161 agaaggctat cgttgatctt cttttcaaga ccaacagaaa ggttaccgtt aagcagttga 5221 aggaggatta cttcaagaag atcgagtgct tcgattctgt tgaaatctct ggagttgagg 5281 atagattcaa cgcttctctt ggaacctacc acgatctttt gaagatcatc aaggataagg 5341 atttccttga taacgaggag aacgaggaca tccttgagga catcgttctt acccttaccc 5401 ttttcgagga tagagagatg atcgaggaga gactcaagac ctacgctcac cttttcgatg 5461 ataaggttat gaagcagttg aagagaagaa gatacaccgg atggggtaga ctttctcgta 5521 agttgatcaa cggaatcaga gataagcagt ctggaaagac catccttgat ttcttgaagt 5581 ctgatggatt cgctaacaga aacttcatgc agcttatcca cgatgattct cttaccttca 5641 aggaggacat ccagaaggct caggtttctg gacagggaga ttctcttcac gagcacatcg 5701 ctaaccttgc tggatctcca gctatcaaga agggaatcct tcagaccgtt aaggttgttg 5761 atgagcttgt taaggttatg ggtagacaca agccagagaa catcgttatc gagatggcta 5821 gagagaacca gaccacccag aagggacaga agaactctcg tgagagaatg aagagaatcg 5881 aggagggaat caaggagctt ggatctcaaa tcttgaagga gcacccagtt gagaacaccc 5941 agcttcagaa cgagaagttg tacctttact accttcagaa cggaagagat atgtacgttg 6001 atcaggagct tgacatcaac agactttctg attacgatgt tgatcacatc gttccacagt 6061 ctttcttgaa ggatgattct atcgataaca aggttcttac ccgttctgat aagaacagag 6121 gaaagtctga taacgttcca tctgaggagg ttgttaagaa gatgaagaac tactggagac 6181 agcttcttaa cgctaagttg atcacccaga gaaagttcga taaccttacc aaggctgaga 6241 gaggaggact ttctgagctt gataaggctg gattcatcaa gagacagctt gttgagacca 6301 gacagatcac caagcacgtt gctcagatcc ttgattctcg tatgaacacc aagtacgatg 6361 agaacgataa gttgatcaga gaggttaagg ttatcacctt gaagtctaag ttggtttctg 6421 atttcagaaa ggatttccag ttctacaagg ttagagagat caacaactac caccacgctc 6481 acgatgctta ccttaacgct gttgttggaa ccgctcttat caagaagtac ccaaagttgg 6541 agtctgagtt cgtttacgga gattacaagg tttacgatgt tagaaagatg atcgctaagt 6601 ctgagcagga gatcggaaag gctaccgcta agtacttctt ctactctaac atcatgaact 6661 tcttcaagac cgagatcacc cttgctaacg gagagatcag aaagagacca cttatcgaga 6721 ccaacggaga gaccggagag atcgtttggg ataagggaag agatttcgct accgttagaa 6781 aggttctttc tatgccacag gttaacatcg ttaagaaaac cgaggttcag accggaggat 6841 tctctaagga gtctatcctt ccaaagagaa actctgataa gttgatcgct agaaagaagg 6901 attgggaccc aaagaagtac ggaggattcg attctccaac cgttgcttac tctgttcttg 6961 ttgttgctaa ggttgagaag ggaaagtcta agaagttgaa gtctgttaag gagcttcttg 7021 gaatcaccat catggagcgt tcttctttcg agaagaaccc aatcgatttc cttgaggcta 7081 agggatacaa ggaggttaag aaggatctta tcatcaagtt gccaaagtac tctcttttcg 7141 agcttgagaa cggaagaaag agaatgcttg cttctgctgg agagcttcag aagggaaacg 7201 agcttgctct tccatctaag tacgttaact tcctttacct tgcttctcac tacgagaagt 7261 tgaagggatc tccagaggat aacgagcaga agcagctttt cgttgagcag cacaagcact 7321 accttgatga gatcatcgag caaatctctg agttctctaa gagagttatc cttgctgatg 7381 ctaaccttga taaggttctt tctgcttaca acaagcacag agataagcca atcagagagc 7441 aggctgagaa catcatccac cttttcaccc ttaccaacct tggtgctcca gctgctttca 7501 agtacttcga taccaccatc gatagaaaaa gatacacctc taccaaggag gttcttgatg 7561 ctacccttat ccaccagtct atcaccggac tttacgagac cagaatcgat ctttctcagc 7621 ttggaggaga taagagacca gctgctacca agaaggctgg acaggctaag aagaagaagt 7681 gactgcagat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 7741 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 7801 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 7861 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 7921 gtcatctatg ttactagatc //