The DNA sequence of small cryptic plasmid pAG20 in Acelobacter aceti was determined at 3064 bp with 51.6% GC pairs. The plasmid encoded a 186 amino acid protein which is important for plasmid replication in Gram-negative bacteria except Escherichia coli. Two 21 bp large direct repeat sequence I and two 13 bp direct repeat sequence 2 were determined in the regulation region upstream from gene encoded Rep protein. Vector pAG24 with kanamycin gene and two deletion derivatives pAG25 and pAG26 without rep gene from plasmid pAG20 were constructed. Plasmid pAG24 was replicated in a broad host range like E coli, Aectobacter pasteurianus, A. aceti, Comanomonas spp., Serratia marcescens, and Shigella spp. (C) 2005 Elsevier Inc. All rights reserved.