Modular Cloning, or MoClo, is an assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIS restriction sites, the user can ligate at least six DNA parts together into a backbone in a one-pot reaction. It is a method based on Golden Gate Assembly, where Type IIS restriction enzymes cleave outside of their recognition site to one side, allowing for removal of those restriction sites from the design. This helps eliminate excess base pairs, or scars, from forming between DNA Parts. However, in order to ligate together properly, MoClo utilizes a set of 4-base pair fusion sites, which remain between parts after ligation, forming 4-base pair scars between DNA parts in the final DNA sequence following ligation of two or more parts.
MoClo utilizes a parallel approach, where all constructs from tier-one(level 0 modules) have restriction sites for BpiI on both sides of the inserts. The vector(also known as "destination vector"), where genes will be added, has an outward-facing BsaI restriction site with a drop-out screening cassette. LacZ is a common screening cassette, where it is replaced by the multigene construct on the destination vector. Each tier-one construct and the vector have different overhangs on them yet complementary to the overhang of the next segment, and this determines the layout of the final multigene construct. Golden Gate Cloning usually starts with level 0 modules. However, if the level 0 module is too large, cloning will start from level -1 fragments, which have to be sequenced, to help cloning the large construct. If starting from level -1 fragments, the level 0 modules do not need to be sequenced again, whereas if starting from level 0 modules, the modules must be sequenced.