ZYCY10P3S2T E.coli ZYCY10P3S2T E.coli Minicircle producer competent cells have been prepared and tested by a modification of the procedure of Kay (Kay MA, He CY, Chen ZY. 2010, Nature Biotechnology). These cells are suitable for the cloning of minicircle-based plasmids. ZYCY10P3S2T E.coli minicircle producer cells are derived from a BW27783 bacterial strain that stably expresses a set of inducible minicricle-assembly enzymes, ФC31 integrase and I-SceI homing endonuclease. This bacterial strain produces purified minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation, making it feasible to use minincircles in place of plasmids in mammalian transgene expression studies. Note: Other competent E. coli strains cannot be substituted for minicircle production as they do not have the appropriate genome modifications to produce minicircles.