Pmir rnl tk reporter plasmid

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.

Nucleotides 3853 - 4363 of the EREG mRNA (accession number: NM_001432.3) were amplified from human gDNA 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. The miRNA expression plasmids were generated by PCR amplification of nucleotides 91,350,658 - 91,351,156 of chromosome 13 (+) for miR-19a-3P and nucleotides 91,350,960 - 91,351,560 of chromosome 13 (+) for miR-19b-3P from human gDNA. Subsequently, the DNA fragments were inserted into the pSG5 vector (Agilent technologies, Ratingen, Germany). Expression plasmid for miR-20b is described elsewhere (8 (link)). The oligonucleotide sequences used for molecular cloning and site directed mutagenesis are shown in Supplementary Table 3.