The Orthodenticle homeobox (Otx) gene is a conserved homeobox gene, crucial for patterning the anterior neural plate of vertebrate embryos. In the invertebrate chordate Ciona intestinalis, Ci-Otx is activated in the anterior neural plate, and dorsal nerve chord by a maternal GATA transcription factor and localized FGF signaling. A minimal 69 bp enhancer (Otx-a) was previously identified in Ciona. It contains 3 GATA binding sites and two ETS sites, which mediate induction by FGF signaling. In order to identify all of the sequences important for enhancer activity we created a library of random synthetic (RS) Otx-a enhancer variants that retain each of the five core recognition sequences, GATA and GGAA, in their native positions. Each of these enhancers variants were attached to a a minimal Fog promoter, GFP coding sequence, andd a unique 30 bp barcode tag, which uniquely identifies each of the different enhancer variants. The fog promoter (Tell significance of barcode tag) was used for these assays since it produces the lowest background expression among the different Ciona promoters tested in previous transgene assays. The resulting library has a complexity of 2.5 x106 unique RS enhancer/barcode tag combinations, and we determined each enhancer/barcode association by deep sequencing. The library was electroporated into thousands of fertilized eggs. Once embryos developed to the late gastrula stage, RNA was extracted, reverse transcribed, amplified by PCR and sequenced to identify transcribed barcodes.</p><p>163,708 unique RNA barcodes exhibited expression, and 21,799 of these produced the same or higher levels of activity than the wild-type enhancer, = 4 normalized reads per million (RPM).