Characterization of AKT3 mRNA Regulation

Nucleotides 1609-2836 of the AKT3 mRNA (accession number: NM_005465.4) were amplified from human genomic DNA by PCR and inserted into pMIR-RNL-TK reporter plasmid (Ambion, Kaufungen, Germany). Mutagenesis of the predicted target site seed sequences of reporter constructs were performed by site directed mutagenesis and with sequence specific primers. The miRNA expression plasmids are described elsewhere [23 (link)]. AKT3 coding sequence (accession number: NM_005465.3) was amplified from human AKT3 gene cDNA clone plasmid (Hölzel Diagnostika Handels, Cologne, Germany) by PCR and inserted into pcDNA3.1 expression plasmid (Thermo Fisher Scientific, Oberhausen, Germany) for in vitro analysis. The primer sequences used for cloning and site directed mutagenesis are shown in Supplemental Table S1.