Gibson assembly cloning

CRY tail chimera rescue vectors were generated by Gibson assembly cloning using NEBuilder HiFi DNA Assembly kit. Briefly, the rescue vector containing Cry1, Cry2, Cry1 7m, or Cry2 7m was linearized by PCR with primers, which also removed the coding sequence for residues 498–606 of Cry1 and Cry1 7m or residues 516–593 of Cry2 and Cry2 7m. The C-terminal Myc tag was left intact. The coding sequences for the tail regions of Cry1 (residues 498–606) and Cry2 (residues 516–593) were amplified by PCR using primers that would generate overlaps between the amplified tail product and the linearized target vectors. PCR products were purified using the QIAquick PCR Purification kit and combined with inserts and linearized target vectors in a 2:1 molar ratio. These combined products were then treated with NEBuilder HiFi DNA Assembly master mix containing an exonuclease, DNA polymerase, and DNA ligase to induce assembly of the final vector. This solution was incubated at 50 °C for 15 min and the products were used to transform DH5α competent cells. Colonies were selected for culturing and miniprep, and insertions were verified by Sanger sequencing.