Abstract: The Tn7 transposon inserts at high frequency in one orientation into a specific site named attTn7. This site is located just downstream of the coding region of glmS. The glmS gene is essential for growth because it is required for cell wall biosynthesis, and therefore also present in most known bacteria. To study and reconstruct Vibrio anguillarum, Edwardsiella tarda and Escherichia coli conveniently, we explored the operation procedure of using Tn7, and found an efficient and convenient method to eliminate the resistance selection marker by Flp/FRT system controlled by promoter pBAD.