Generating Chimeric Cr2c-Cyclop1 Constructs

Based on the pGEMHE-Cr2c-Cyclop1 construct from our previous experiments [8 (link)], three chimeras were generated by exchanging DNA fragments between Cop5 and Cr2c-Cyclop1 with certain restriction sites. The vector pGEMHE-Cr2c-Cyclop1 was digested with 5′-BamHI and 3′-NcoI. For chimeras 1 and 2, the Cop5 part were amplified with 5′-BamHI and 3′-PpuMI restriction sites by PCR, and the amplified fragments were digested by the two enzymes correspondingly. Meanwhile, the other missing DNA fragment from Cr2c-Cyclop1 part was PCR-amplified and digested by 5′-PpuMI and 3′-NcoI. Finally, two fragments from Cop5 and Cr2c-Cyclop1 were inserted to the BamHI/NcoI cut vector in one reaction. To clone chimera 3, the DNA fragment was amplified from Cop5, digested with BamHI/NcoI and directly ligated to the same vector.
All cloned chimeras were confirmed by complete DNA sequencing. To generate linearized DNA, plasmids were digested by NheI. cRNA was then generated after in vitro transcription by the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies).