Improvement of the generation of pri-smiRNAs for knockdown lines. (A) Backbone of the endogenous pri-miR159a driven by the strong 35S promoter. The expression cassette flanked by the HindIII and EcoRI sites of the pUC19 vector was moved into the binary vector pGPTV-BAR for the generation of transgenic lines. (B) Standard procedure to create a pri-smiRNA by exchanging miR159 with smiRNA and miR159* with smiRNA* sequences, respectively, by a two-step procedure involving overlap-extension PCRs. (C) "Easy cloning vector" (ECV) procedure that allows the generation of any pri-smiRNA by a one-step PCR-cloning procedure due to the introduction of restriction sites. Primers required for each procedure are indicated by arrows. Vector primers are in black. The sequences of the introduced restriction sites are given as inserts.