Lr recombination clonase

Pineapple genomic DNA was used to amplify 5058 bp of complete AcSWC6 gene from start codon and without stop codon using the primers listed in Supplementary Table S2. The amplified AcSWC6 was then moved to the vector pENTR/D-TOPO (Invitrogen). The positive clones were then recombined with the destination vector pGWB505 using LR recombination clonase (Invitrogen, Carlsbad, CA, USA) [9 (link)]. The final cassette was transformed into GV3101 strain of Agrobacterium tumefaciens and used for transformation of Atswc6 and Col-0 using the floral dip method [46 (link)].

The full-length CDS of TaHSP23.9 was amplified by specific primers that listed in Supplementary Table S1. The PrimeSTAR HS DNA Polymerase (Takara, Dalian, China) was used for amplification, and the expected PCR fragment was cloned into pDONR/Zeo entry vector by BP recombination clonase (Invitrogen, Carlsbad, CA, United States). After the sequence was 100% matched the sequence published on the website², the TaHSP23.9 was cloned into pBIB-BASTA-35S-GWR destination vector by LR recombination clonase (Invitrogen, Carlsbad, CA, United States). The recombinant plasmid pBIB-BASTA-35S-TaHSP23.9 was introduced into the Agrobacterium tumefaciens strain GV3101. Transformation of Arabidopsis thaliana was performed by the floral dip method (Clough and Bent, 1998 (link)). The homozygous lines with single insertion were obtained from transformants that produced 100% BASTA-resistant progenies in the T₃ generation, and three independent homozygous 35S-TaHSP23.9 lines were selected for further experiments.

Mouse KCNH6 and Munc-18-1 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy and were verified by DNA sequencing. The sequences of the primers used are listed in Supplementary Table 2. These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG vector (Invitrogen) or mCherry-C1 vector (Clontech) as described previously [24 (link)]. Insulin-EGFP was generated as described previously [25 (link)]. To generate recombinant adenoviruses, KCNH6 WT and KCNH6 R246A/T248A/L250A (3A) mutant were inserted into pENTR-3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen), which co-produces red fluorescent protein (Cherry) to allow identification of transfected cells. To express an exogenous protein, HEK293A cells were transfected with the plasmids using Lipofectamine 3000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses.