Tester construct designs and sequences.

A. Guide sequences and their sources for each gene targeted in test 4[sh], 8[sh], 12[sh], 16[sh] clusters (top) and 39 nucleotide-long sequences selected as spacers to separate individual hairpins (bottom). Spacer sequences are derived from well-characterized Drosophila microRNA genes as indicated. B. 4[sh] test cluster design. Sequences that make up each short hairpin (5’ flank, passenger, loop, guide and 3’ flank), concatenated short-hairpin sequences, and the full clusters, including the spacer sequences between individual short hairpins are provided. C. Primers for 4 sequential PCR reactions used to synthesize the 4[sh] test clusters. D. Additional 4[sh] clusters designed to append to tester-1 to generate 8[sh], 12[sh] and 16[sh] test clusters. (XLSX)