To obtain DNA fragments containing the ESG1 gene, we screened the bacterial artificial chromosome (BAC) DNA pool by PCR using primer pairs that would only amplify the real gene, not the pseudogenes. We obtained two independent, but overlapping BAC clones. Southern blot analyses and sequencing demonstrated that these clones contained a sequence exhibiting complete identity with ESG1 cDNA that was interrupted by two putative introns (Figure 2A). The two intron sequences begin with GT and terminate with AG, fulfilling the GT-AG rule of exon-intron junctions [19]. The 5' flanking region of this DNA fragment exhibited strong promoter/enhancer activity by luciferase reporter assays in undifferentiated ES cells, but not in somatic cells (Figure 3). The same fragment showed much weaker activity after induction of differentiation by retinoic acid. We concluded that this sequence is the ESG1 gene.