Nucleic Acids Research, 2010, Vol. 38, No. 8 2633 Figure 5. Insertion of an antibiotic resistance gene and terminator swapping through a three-way In-Fusion Assembly. (a) T9002, B0032/kanR, and B0011 plasmids are shown with the forward and reverse primers for PCR. B0011 is ampliﬁed with the vector and both T9002 and B0032/kanR are ampliﬁed as inserts. (b) Detailed schematic of the assembly strategy with the forward and reverse primers. The entire T9002 circuit is ampliﬁed upstream of B0010 (Part A) with homology to connect an RBS and kanR gene downstream (Part B). To avoid the use of repeated transcriptional terminators (as in T9002 but not shown in the ﬁgure), a B0011 terminator (Part C) was placed downstream of kanR. (c) Colony PCR results show that 4 out of 12 colonies were positive for the correctly assembled construct (correct size indicated by arrow). A negative control was not necessary since only a 3-kb fragment would indicate a successful colony.