We have developed an Escherichia coli expression vector that is particularly useful for construction and production of fusion proteins. Based on the synthetic biology pSB1C3 platform, the resulting vector offers a combination of useful features: the strong T7 promoter combined with lac operator, OmpA signal sequence, a selection of cloning sites located at convenient positions and a 3'-terminal His-10 tag. Each of these regions is flanked by a restriction site that allows for easy vector modification, including removal of the signal sequence without perturbation of the reading frame. All the elements were assembled by stepwise addition of three cassettes for which the design was made de nova. To prove the efficiency of the new vector, named pMD204, we successfully produced a cysteine proteinase inhibitor variant in the periplasm and in the cytoplasm of E. coli, in both cases as a soluble and active protein. (C) 2008 Elsevier Inc. All rights reserved.