Extended Data Fig. 3: Insulation effects of synthetic CTCF binding sites.

a, Additive insulation by synthetic CBS from boundary regions. Left top, compositions of one 139bp-CBS that was synthesized; Left bottom, tandemly arrayed 139bp-CBSs tested for insulator activity. Right, normalized Sox2-eGFP expression of clones with the tandemly arrayed 139bp-CBSs inserted between the Sox2 gene and its super-enhancer. Blue, CBS core motifs were in forward orientation; Red, CBS core motifs were in reverse orientation. Insertions were on the CAST allele only. n = 3, unpaired t-test, two-tailed. Data are mean ± sd. b, Insulation effects of PCR cloned large size CBSs (1–4 kb) and the synthesized 139bp-CBSs that contain the same CTCF motifs. (n = 12, paired t-test, two-tailed, ***P = 0.0007.). c, CTCF binding strength at selected boundary sites and non-boundary sites in mouse ES cells. ChIP-seq signals of CTCF are shown in 2-kb window. d, ChIP-seq of CTCF and Rad21 in clones with the insertion of six (nBd-syn6) or fifteen (nBd-syn15) 139-bp CBSs obtained from non-boundary regions. ChIP-seq reads were mapped to a customized mm10 genome that included the inserted sequence at the target site. Insertion position is highlighted in the red box.