Nucleic Acids Research, 2010, Vol. 38, No. 8 2629 Figure 3. Assembly of two basic parts. (a) R0011 and E0240 plasmids are shown with the forward and reverse primers for PCR. R0011 is ampliﬁed with the vector and E0240 is ampliﬁed as the insert. (b) Detailed schematic of the assembly strategy with the forward and reverse primers. The R0011-ampliﬁed PCR product has an additional scar sequence and RBS that has homology to the E0240-ampliﬁed PCR product. (c) Primer design schematic to test the eﬀect of homology length on success and mutation rates. The desired sequence for R0011, scar sequence, and E0240 is shown. For each primer pair, forward primers are shown as right arrows, reverse primers as left arrows, and the region of homology between primers is boxed. Homology and primer lengths are shown where bolded numbers highlight diﬀerences between primer pairs. (d) Primer design schematic to test the eﬀect of primer length on success and mutation rates. Details are similar to that described in (c).