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Fig. 4 Design of synthetic enhancers from short multiplexed sequences. (a) We generated concatemers containing five copies of each of the six multiplexed sequences. Three of these (5x2CD12/epidermis, 5x2GH12/notochord, 5x3CD05/spinal cord) drove more intense expression in the same tissue as the original 15-bp sequence. The fourth construct, 5x2GH08/brain, did not produce any detectable expression, suggesting that the XhoI-flanking sequence may be important. White arrows mark the epidermis, notochord, and spinal cord, respectively. (b) A second version of the 2GH08/brain concatemer contained five tandem repeats of the XhoI flanking region with the 2GH08(L9) functional sequence that was identified by the 9-bp dissection experiments. This arrangement produced a strong regulatory element that drives consistent expression to the forebrain and otic lateral line at 24 and 48 hpf. White arrows mark the forebrain and otic lateral line patterns. All pictures are of 24 hpf embryos.