Experimental system and constructs used in this study. (A) Structure of the human β-globin locus with the locations of the two replicators (Rep-P and Rep-I) and the asymmetric purine:pyrimidine (AG) region marked. LCR, locus control region; IR, initiation region. (B) Transgene constructs. LCR, a mini-LCR sequence that includes the DNase I-hypersensitive sites 4, 3, and 2 from the human β-globin locus control region; Pro, human β-globin promoter; EGFP, enhanced green fluorescence protein gene. (C) All transgene constructs flanked by a pair of inverted loxP sites, L1 and 1L, were inserted into a late-replicating RL4 site of the murine chromosome 15 by recombinase-mediated cassette exchange (RMCE). The insertion replaces the hygromycin-thymidine kinase (Hyg-TK) hybrid selection marker. (D) Wild-type and mutant sequences of the AG region of Rep-P used in this study. Nucleotides shown on a white background are mutations. The CG (red) below the sequences indicates the CpG dinucleotide created in the Rep-PAG2 mutation.