Figure 4

Amplified insert Assembly. The insert part is amplified from its plasmid using high-fidelity PCR and the purified PCR product is digested with XbaI, PstI, and DpnI. Meanwhile the vector part's plasmid is digested with SpeI and PstI and phosphatased. The products of these two digestions are then ligated and the circular product is transformed into cells. This assembly can easily be done in reverse manner using the promoter as the insert and leaving the coding sequence in its vector, thus providing additional flexibility.