Additional Supporting Information may be found in the online version of this article.

DVDY_22728_sm_SuppFig1.tif5317KSupporting Information Figure 1. RPS elements that are shared among functionally related enhancers are not found in any common order or orientation within the different enhancers. Shown are relaxed D. melanogaster genomic DNA EvoPrints (12 drosophilids) of the functionally related cas-6, cg7229-5 and grh-15 late temporal NB enhancers. Highlighted with different colors are 8 RPS elements of 6 bp or greater in length that are present in each of the three enhancers. Note, many of the RPS elements overlap other elements but in different and arrangements within the enhancer CSBs.
DVDY_22728_sm_SuppFig2.tif3870KSupporting Information Figure 2. Primary and shadow enhancer CSBs share common balanced sequence elements. Shown are relaxed D. melanogaster genomic DNA EvoPrints (12 drosophilids) of functionally related enhancers that regulate vnd gene expression in overlapping expression patterns within the embryonic neurogenic ectoderm (described by Hong et al., 2008; boxed sequences indicated enhancer-reported tested DNA). Highlighted CSB sequences represent shared ≥6-bp unique or repeat sequence elements between the primary and shadow enhancer CSCs. Gray highlighted sequences are present once in the vnd primary enhancer CSC (note; when uniquely shared elements overlap repeat sequences, the repeat-ratio highlight color is shown). Green indicates balanced repeat numbers between the two CSCs; and yellow highlights repeats that differ in frequency by just one copy. When repeat elements overlap, the balance-ratio highlight of the longer repeat is shown; when two repeats of equal size overlap, the more balanced repeat highlight is shown.
DVDY_22728_sm_SuppFig3.tif3590KSupporting Information Figure 3. cis-Decoder analysis of sequences flanking P{Gal4} insertions identifies a flanking enhancer that regulates the P-element transgene. A: A linear cartoon of the CG32264 predicted gene locus on chromosome 3L. Dark-blue boxes indicate ORF containing exons, the P{Gal4}c492a (Armstrong and Kaiser, 1997) integration site is indicated as a red-colored triangle, and the position of the cg32264-76 CSC is highlighted as a light-blue box. B: A relaxed EvoPrint of the cg32264-76 CSC genomic region (boxed sequence indicates the enhancer-reported tested DNA). CSB sequences that span repeat elements are highlighted in light purple color. One-on-one CSB alignments of the cg32264-76 CSC, utilizing cis-Decoder, identified 14 distinct repeat elements that are ≥6 bp. A total of 31 repeat elements cover 57.46% of the conserved sequences. Highlighted with different colored bases are underlined sequences that correspond to the core DNA-binding sites of different transcription factors (homeodomain, ATTA-red; POU domain, ATGCAAAT-green; bHLH, CANNTG-brown: Hunchback/Castor, TTTTT/AT-blue; and PBX sites, TGAT-teal). C: In the adult brain, the cg32264-76 CSC enhancer-Gal4 driver transgene activates GFP-CD8 tagged transgene expression the central brain, sub-esophageal ganglion, and in optic lobe neurons. Shown is a confocal, optical section of an adult brain (frontal view).
DVDY_22728_sm_SuppFig4.tif410KSupporting Information Figure 4. Bullfinch, a site-specific integration vector, consists of a modified pCa4B vector (Markstein et al., 2008) with an inserted polylinker site (A. Kuzin), minimum Heat shock protein 70 promoter (from the pRed H-Stinger vector; Barolo et al., 2004), Gal4 ORF (from S. cerevisiae), and SV40 3′UTR (from the pRed H-Stinger vector; Barolo et al., 2004).
DVDY_22728_sm_SuppTab1.pdf106KSupporting Information Table 1. Conserved sequence clusters: Location and cloning primers

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