All 14 essential genes of chrIII were clustered and reordered, and transcription of the genes was tested. We designed and constructed three types of reorganized essential genes into three different plasmids. For the genomic direction (GD) plasmid, the essential genes were arranged according to their natural chromosomal positions and orientations. For the same direction (SD) plasmid, the essential genes were arrayed in the same positions as GD plasmid, but all genes were assembled in the same direction (SD). For the random direction (RD) plasmid, the essential genes were modularly assembled according to the combined length of three individual genes and thus displayed disorderly. The synIII strains bearing the GD/SD/RD plasmids were phenotypically normal despite of the double expression of 14 essential genes. Overall design: PCRTags were used to distinguish the transcription of wild-type genes from the transcription of synthetic genes.