The Transcription Regulatory Element Database (Jiang et al. 2007) was used to identify the ~2 Kb sequence surrounding the transcriptional start site of SEPP1, promoter ID #34663 (1770 bp upstream of start site, 300 bp downstream) and used as an electronic template to generate promoter constructs. A 1.9 Kb sequence was amplified from human genomic DNA by PCR using the primer pair 5′-TAGGTACCCCAGTTCTTTCCGGTGTTCA-3′ and 5′-TACTCGAGCGCACTGGGAACTTCACCTA-3′. The PCR product was digested with XhoI and SstI and cloned into the pGL4.21 luciferase reporter vector. This construct is referred to as −1652 to +247 and was utilized as template DNA in subsequent PCR reactions used to synthesize smaller fragments of the SEPP1 promoter region of interest. A HindIII digestion of the −1652 to +247 construct generated −1652 to −385 and −391 to +247 promoter fragments. The fragments were cloned into the pGL4.21 vector following HindIII digestion. Due to the use of the HindIII site in the pGL4.21 vector, the −391 to +247 fragment was only subcloned in the reverse orientation, and despite several attempts, no colonies were obtained with this fragment in the
