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Synthetic biology circuits are often constructed with multiple gene expression units assembled in close proximity, and can be used to perform complex functions in embryonic stem cells (ESCs). However, mutual interference between transcriptional units has not been well studied in mouse ESCs. To assess the efficiency of insulators at suppressing promoter interference in mouse ESCs, we utilized an evaluation scheme, in which a tunable TRE promoter is connected to a constant Nanog promoter. Chicken hypersensitive site 4 (cHS4) insulator, w