刘耀光_多靶点pCRISPR载体(单子叶和双子叶植物用)使用方法2015-4-14(new)(1)

tggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaaataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtca (19-20 bp target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

AtU3d-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAataagctt atgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone）

PCR product of AtU3d-sgRNA (284 bp, 254 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcA(19-20 bp target)GTTTTAGAGCTA GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGT GGATGGNNNNNNNCGAGACCCACGCT

PCR product of AtU6-1-sgRNA (487 bp, 457 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAAGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAGACGAAGTAGTGATTG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

AtU6-29-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattggtagagtcgactaagagattgAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)PCR product of AtU6-29-sgRNA (502 bp, 472 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattggtagagtcgactaagagattG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

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