Pet 28b ggcad q467v t470a

For construction of GgCL expression plasmids, the LOC418544 (NCBI GeneID: 418544) CDS sequence (XM_015300896) inserted into pcDNA3.1+/C-(K)DYK vector was purchased from GenScript (USA Inc.). The sequence was then amplified using CBSL_Fw, CBSL_Rev primers (native GgCL) or CBSL_Fw, CBSL_short_Rev (truncated GgCL) by PCR, using Phusion DNA polymerase, and inserted into pET-28b expression vector at NdeI/XhoI sites, generating respectively pET-28b-nativeGgCL and pET-28b-truncatedGgCL. Details of the designed primers are reported in supplementary Table 1. A first transformation of the constructs into E. coli XL1Blue strain by electroporation was performed for plasmid amplification. Plasmids were extracted by alkaline lysis and transformed into E. coli BL21 Codon Plus strain by electroporation. For construction of GgCAD expression plasmids, GgCAD wild-type sequence (NCBI GeneID: 426184) and mutated sequences (Q467V, T470A, Q467V-T470A) inserted into pET-28b expression vector were purchased from GenScript (USA Inc.), generating respectively pET-28b-GgCAD, pET-28b-GgCAD_Q467V, pET-28b-GgCAD_T470A, pET-28b-GgCAD_Q467V-T470A. The constructs were transformed directly into E. coli BL21 Codon Plus by electroporation. The authenticity of all constructs was verified by sequence analysis.