Regulatory elements controlling gene transcription are scattered across the genome, yet it remains poorly understood how their precise positioning shapes the regulatory landscape. We previously developed a method to relocate a regulatory element to thousands of positions within a genomic locus and measure position-dependent effects on gene regulation. Here, we apply this approach to systematically probe the role and position dependence of CTCF binding sites (CBSs), which are thought to modulate communication between distal regulatory elements. We focused on the mouse Sox2 locus in embryonic stem cells, where Sox2 is activated by a potent distal enhancer. First, we show that a CBS naturally located upstream of the Sox2 promoter confers strong orientation-dependent promoter activation throughout the gene-enhancer interval. Second, insertion of CBSs alone within this interval consistently reduces Sox2 expression in an orientation-dependent manner, suggesting polarity in loop extrusion. Third, two native CBSs within and downstream of the enhancer subtly help confine the enhancer's activation range. Together, these results illuminate how CBSs interact with the detailed regulatory landscape of a genomic locus.