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. 2012 Jul 19;7(7):e41504. doi: 10.1371/journal.pone.0041504

Figure 1. Design and generation of plasmids for the overexpression of microRNA* species.

Figure 1

(A) Design of the stem-loop miRNA precursor. The mature sequences of miRNA* species were incorporated into the 3′ arm of the precursor, whereas the complementary sequences of the miRNA* species were incorporated into the 5′ arm. Note that the complementary sequences could either be completely complementary to the miRNA* species or could be modified by mutating several bases in its seed region, which were not complementary to the miRNA* species. (B) The designed hsa-miR-146b-3p stem-loop precursor. The red characters in the 3′ arm correspond with the sequences of hsa-miR-146b-3p. The 5′ arm sequences are the modified complementary sequences of hsa-miR-146b-3p, and the sequences above are hsa-miR-146b-5p, with blue characters denoting seed sequences and solid lines between them indicating the similarity of the two sequences. The dotted lines show the modified sites in the 5′ arm sequences and their corresponding nucleotides in hsa-miR-146b-3p sequences. (C) The uppermost oligo is the DNA insert of hsa-miR-146b-3p precursor obtained by annealing two single stranded DNA oligos with BamH1 and EcoR1 sticky ends. The DNA insert was incorporated into plvx-shRNA2 between the recognition sites for the restriction enzymes BamH1 and EcoR1. The representation of the vector was modified from an illustration in the instructions provided with the product.