Enhancer and control regions were PCR-amplified using KOD plus polymerase (TOYOBO) from HEK-293T gDNA, digested with BamHI and SalI (TAKARA BIO), and purified using the E-Gel® SizeSelect™ system (Life Technologies). Five μl of purified PCR products were ligated with 100 ng of the BamHI- and SalI-digested modified pGL4.10EF1α and pGL4.10 plasmids using Ligation-high (TOYOBO), and transformed into DH5α competent cells (TOYOBO). Correct insertion of the PCR products into the plasmids was checked by colony PCR. Vectors were purified using the QIAGEN Plasmid Plus 96 Miniprep Kit (QIAGEN).
