Figure 4. Creation of seamless constructs by Hot Fusion.

(A) A schematic diagram for the creation of a seamless construct. The vector was linearized by SphI digestion (leaving CATG 3′ overhangs), heat-inactivated and directly used for cloning. The PCR products contain the overlapping sequences at each end (excluding the CATG overhang). The ribosome binding site is underlined. (B) Examples of the transformation plates. Blue colonies contain the parental vector and white colonies contain potential positive clones. The negative control is shown as plate a (unpurified digested vector without added PCR product). Plates, b and c are two examples of transformation plates containing potential positive clones. (C) PCR screening results of 8 colonies for each construct (each plate). Eight PCR products of each construct were loaded every other lane on agarose gels by multi channel pipette.