Figure 1.

Experimental design and validation. A) Schematic of the high throughput screen for ADs. Cells containing a GFP reporter driven by a synthetic Gcn4-dependent promoter were transformed with libraries of random 30-mers fused to the N-terminus of the Gcn4 DNA binding domain. Cells with Gcn4-AD function were enriched by growth in 3-AT followed by FACS. DNA from the libraries before 3-AT selection and FACS (background library) and from the four GFP-containing bins were sequenced. The AD-negative set was created by removing sequences found in bins 1-4 from the background library. TES: ADH1 terminator; pA: poly-A site. B) Plots show the number of cells vs relative fluorescence intensities from FACS analysis of cultures with WT Gcn4, the enriched library, and no Gcn4. Vertical lines show gates used for binning AD-containing cells. C) Experimental validation of enrichment scores on 18 AD sequences versus GFP expression in the reporter strain. Individual clones were assayed by FACS and the mean fluorescence of the cell population is shown. See Table S1 and Fig S1.