synthetic binding site of shRNA 122 (perfect) KD96:%
perfect binding site of liver specific shRNA 122 in the context of the tuning construct. This binding side leads to a knockdown percentage of 96% percent if introduced into the 3'UTR of the gene of interest (GOI). It can be either co-transfected with a synthetic miRNA or transfected into liver cell lines (i.e. Huh7).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
characterization of part binding sites (BBa_K337055)
Binding sites for miR122 were experimentally characterized by cloning them into psiCHECK-2 backbone (Promega). Dual luciferase assay was conducted using this time Renilla Luciferase as a reporter and the other luciferase as a reference for normalization. Figure 2 shows again a broad range of regulation depending on binding site sequence properties. BBa_K337055 (perfect BS) is the construct with the perfect binding site and leads to an knock-down percentage of 96%. BBa_K337056 (mut 7) is an imperfect binding site which leads to a knockdown percentage of 64%. BBa_K337057(mut 111) is an imperfect binding site with a knockdown efficiency of 24%. All this contributes to a real tuning effect by introducing binding sites with introduced mismatches following the rational design protocol.