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Design of additional shRT5 mutants varying in loop size and stem length. The shRT5 with a 19 bp stem and a 5-nt loop (19/5) was used as backbone for this design. The shRNA stem length was reduced/extended from the bottom of the hairpin, resulting in shRT5 variants 15/5–23/5. In addition, shRNA terminal loops ranging in size from 3 to 8 nt were designed (19/3–19/8) and the loop sequence was reversed (19/5R). The original shRT5 21/5A variant was also included.
