Figure 5

An example of chimera construction with a short (105 bp) DNA fragment replaced by a synthesized insert of 99 bp (included in two primers). (A) A region of α7 nAChR subunit sequence with a 105 bp fragment (blue) to be replaced. (B) Amino acid sequence alignment of human α7 nAChR and corresponding region of Aplysia californica AChBP. Segments to be replaced are colored with blue (35 codons) for α7 nAChR sequence and red (33 codons) for AChBP sequence. (C) Target chimera construct with the human nAChR α7 subunit sequence (black) and a substituted 99 bp DNA fragment (red) from the mammalian codon-optimized homologous sequence of the AChBP. Two colored arrows indicate two primers with a 15 bp overlapping region (highlighted). Note that the entire insert of the 99 bp fragment is included in the two primers. Thus, there is no need to amplify the insert. The length of each primer is 81 bases for the forward primer and 76 bases for the reverse primer.