Pgl3 basica vector

For the ENC1-P luciferase reporter gene, the 1851-bp region of the ENC1 promoter was subcloned into the XhoI and HindIII sites of the pGL3-Basica vector (Promega Corporation, Madison, WI, USA). To generate the ENC1 promoter enhancer (PE) luciferase reporter genes, the 1899-bp region of the ENC1-E1 enhancer, the 1838-bp region of the ENC1-E2 enhancer, the 1533-bp region of the ENC1-E3 enhancer, and the 1999-bp region of the ENC1-E4 enhancer were subcloned into ENC1. The luciferase reporter genes ENC1-E1, ENC1-E2, ENC1-E3, and ENC1-E4 plasmids were generated using 4-Promoter luciferase reporter gene with the SalI/BamHI restriction enzyme site. To confirm the TCF4 binding sites on the ENC1-E2 enhancer, a 241 bp region containing the wild-type (WT) TCF4 motif-binding sequence (from chr5: 74687892-74684243) was inserted into the SalI and BamHI sites of the pGL4 vector (downstream of the luciferase) and the minimal promoter (Promega) to produce the TCF4 luciferase reporter gene (pGL4-WT). To generate the pGL4-mutant (Mut) plasmid, mutant TCF4 binding sites from GTGGTGGTTT to GTGAACGTT were constructed by PCR-based site-directed mutagenesis.