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We utilized a bacterial selection system to isolate Engrailed variants from a randomized library that are compatible with each of the 64 possible 3’ triplet sites (i.e. TAANNN). The DNA-binding specificity of 151 representative HD variants from these populations was subsequently characterized. Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs. These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in their specificity. The identified determinates expand our understanding of HD recognition. Homeodomains where selected against each of the 64 possible 3' triplet sites (ie: TAANNN). The DNA-binding specificity of 151 representative HD variants from these populations was subsequently characterized. 7 novel determinants, when grafted into 3 different HD backbones. 5' speicifity was changed for 3 novel determinants.

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