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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all <t>mTurquoise2</t> reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.
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(A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all mTurquoise2 reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.

Journal: bioRxiv

Article Title: Effects of CTCF on the regulatory landscape of the mouse Sox2 locus

doi: 10.64898/2026.03.16.711995

Figure Lengend Snippet: (A) Overview of the Sox2 locus. (Top to bottom) RCMC contact maps (1-kb resolution) in mESCs from ; positions of SCR, eGFP/mCherry tagged Sox2 alleles and launch pad (–116 kb from SCR on 129S1 allele) containing SB with a hygromycin phosphotransferase-thymidine kinase (HyTK) recombination cassette flanked by FRT and F3 recombination sites. Position and orientation of endogenous CTCF binding sites depicted as grey triangles (B) Schematic representation of different Sox2 reporters; all mTurquoise2 reporters are driven by Sox2 promoter and 5’UTR, followed by the SV40 polyadenylation signal (PAS). 1xCBS_Fw_Sox2P reporter carries 221 bp upstream of the Sox2 promoter containing the Sox2 promoter proximal CTCF motif (forward). 3xCBS_Fw_Sox2P reporters carry 329 bp containing three strong forward oriented CTCF binding motifs upstream of the Sox2 promoter. 3xCBS_Rv_Sox2P reporters carry the same three CTCF binding motifs in reverse orientation.

Article Snippet: The Sox2 promoter fragment was then excised with XbaI and SalI and cloned upstream of mTurquoise2 (Addgene #118617) to generate a Sox2P–mTurquoise2 reporter, followed by insertion of an SV40 polyadenylation signal 3′ of the fluorophore.

Techniques: Binding Assay