The number of DNA segments that can be assembled in one reaction is dependent on the length and sequence of the fragments. Gibson Assembly has been used to efficiently assemble up to twelve 0.4 kb inserts into a vector at one time. However, we recommend the assembly of five or fewer inserts into a vector in one reaction in order to produce a clone with the correct insert. A strategy involving sequential assembly can be used if all of the fragments cannot be assembled in a single reaction. Alternatively, consider Golden Gate Assembly for assemblies of >6 fragments.