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For large scale vector construction:

If different target fragments are separately inserted into one vector, the “OL” regions with the vector should be designed overhanging at the 5’-ends of insert replication primers “IF/IR”, so that the linear vector from one pair of replication primers “VF/VR” is sufficient to connect with each insert (shown as a). On the contrary, when one target fragment is inserted into different vectors, the intact insert is amplified by one pair of primers “IF/IR” and the “OL” regions with the insert should be designed overhanging at the 5’-ends of linear vector replication primers “VF/VR” (displayed as b).










e.g., "G50"

e.g., "G50"