Level -1 fragments are used to help cloning large level 0 modules. To clone level -1 fragments, blunt-end cloning with restriction ligation can be used. The vector used in cloning level -1 fragments cannot contain Type IIS restriction site BpiI that is used for the following assembly step. Moreover, the vector should also have a different selection marker from the destination vector in next assembly step, for example, if spectinomycin resistance is used in level 0 modules, level -1 fragments should have another antibiotic resistance like ampicillin.