Limulus Factor C cDNA Design

Limulus Factor C is a relatively large protein (1020 amino acids), so the synthesis of its coding sequence presented a challenge. We determined that instead of trying to have the entire construct synthesized as one piece it would be more cost effective to build it in pieces. The coding sequence for Limulus Clotting Factor C (LFC) was designed as four contiguous blocks to be joined together via overlap extension PCR. Three of the four blocks were 800 bp long; the fourth block was 660 bp long. We designed primers for overlap extension PCR to join the g-blocks together into the final Factor C cDNA sequence. Primers were designed in both forward and reverse complement to the top and bottom strands of the blocks. For example, the forward primer of Block 2 was created with a few base pairs from Block 1 5’ top strand end. These base pairs were added to the beginning of the 5’ top strand to start amplification of Block 2’s top strand. The reverse complement primer for Block 1 was created by taking a few base pairs from the end of the 3’ bottom strand of Block 1 and adding it to the bottom strand of Block 2. The goal of creating these primers is to create overhangs from the previous block to bind to the next block. During amplification, the blocks would then be joined and extend the 5’ top strand and 3’ bottom strand of each block. Once all four blocks and a total of 8 primers were created, we began PCR amplification of Blocks 1 and 2 and then amplified Blocks 3 and 4. The fusion of the coupled blocks would then be fused together to make the final product, LFC Blocks 1-4 via overlap extension PCR.