Level 2 vectors have two inverted BpiI sites from the insertion of level 1 modules. The upstream fusion site is compatible to a gene cloned in level 1 vector while the downstream fusion site has a universal sequence. Each cloning allows 2-6 genes to be inserted in the same vector.
Adding more genes in one cloning step is not recommended, for this would result in incorrect constructs. On one hand, this can induce more restriction sites in the construct, where this open construct allows additional genes be added. On the other hand, this can also eliminate restriction sites, where this close construct stop the further addition of genes.
Therefore, constructs of more than six genes need successive cloning steps, which requires end-linkers containing BsaI or BsmBI internal restriction sites and blue or purple markers. Each cloning step needs to alternate the restriction site and the marker. Furthermore, two restriction enzymes are needed, where BpiI is used for releasing level 1 modules from level 1 constructs and BsaI/BsmBI is for digesting and opening the recipient level 2-n plasmid. When screening, the correct colonies should alternate from blue to purple every cloning step, but if a "closed" end-linker is used, the colonies will be white.