Cloning of miRNA Expression Constructs

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.