Principle of suboptimization
The enhancer consists of low-affinity TF binding sites in a suboptimal syntax, in terms of flanking nucleotides and spacing between the sites. Optimizing the TF binding affinities can lead to ectopic expression.
In sea squirt, 15 synthetic enhancers consisting of 2 ETS binding sites and a ZicL binding site. Only 2 show activities. Further manipulation confirm key roles for binding site orientation, spacing and flanking nucleotides.
Activity of enhancers depend on binding affinities of sites, flanking bases, site arrangement (orientation and spacing). For a particular enhancer, he desired activity level is not necessarily the strongest.