2628 Nucleic Acids Research, 2010, Vol. 38, No. 8 Figure 2. General strategies for In-Fusion BioBrick assembly. There are two general strategies for In-Fusion assembly on pSB1A2 plasmids depending on which BioBrick is PCR-ampliﬁed with the vector. The ﬁrst strategy PCR-ampliﬁes the upstream part by itself and the downstream part with the vector (a). The second strategy PCR-ampliﬁes the upstream part with the vector and the downstream part by itself (b). Each strategy requires four primers, where two are speciﬁc to the parts to assemble and two are general vector primers. The vector primers are described in the ‘Materials and Methods’ section and can either be speciﬁc to the preﬁx or suﬃx. When Part B is ampliﬁed with the vector, preﬁx primers are used. When Part A is ampliﬁed with the vector, suﬃx primers are used. All other details are described in Figure 1b.