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Engineering of amiRNAs.
Site-directed mutagenesis on precursors of endogenous miRNAs was performed using overlapping PCR. Oligonucleotide primers I to IV were used to replace miRNA and miRNA* regions (blue) with artificial sequences (red). Primers A and B were based on template plasmid sequence. Regeneration of functional miRNA precursors was achieved by combining PCR products A-IV, II-III, and I-B in a single reaction with primers A and B.
