The 148-bp EcoRI/XhoI fragment containing the herpes simplex virus thymidine kinase (tk) promoter from pTKβ (Clontech, Mountain View, CA, USA) was ligated into the EcoRI/XhoI sites of the pBluescript® II KS (-) vector (Stratagene, La Jolla, CA, USA) to generate pBKS-Ptk. Two oligonucleotides, PPRE sense (5′-TCGACAGGGGACCAGGACAAAGGTCACGTTCGGGAC-3′) and PPRE antisense (5′-TCGAGTCCCGAACGTGACCTTTGTCCTGGTCCCCTG-3′) containing the PPRE sequence of the acyl-CoA oxidase gene and carrying the SalI and XhoI restriction sites at the ends, were annealed and ligated to form two (72 bp), three (108 bp), four (144 bp), five (180 bp), six (216 bp), seven (252 bp), and eight (288 bp) tandem repeats of PPRE. The PPRE tandem repeats were then filled in and inserted into the blunted XbaI site of the pBKS-Ptk vector to generate pBKS-PPRE2x-Ptk, pBKS-PPRE3x-Ptk, pBKS-PPRE4x-Ptk, pBKS-PPRE5x-Ptk, pBKS-PPRE6x-Ptk, pBKS-PPRE7x-Ptk, and pBKS-PPRE8x-Ptk. The SmaI/BglII fragments containing the tk promoter and/or PPRE repeats from aforementioned constructs were then subcloned into the SmaI/BglII sites of the pGL2-Basic vector (Promega, Madison, WI, USA) to generate pGL-PPRE0x-Ptk, pGL-PPRE2x-Ptk, pGL-PPRE3x-Ptk, pGL-PPRE4x-Ptk, pGL-PPRE5x-Ptk, pGL-PPRE6x-Ptk, pGL-PPRE7x-Ptk, and pGL-PPRE8x-Ptk (Figure 1(A)).
