In fusion kit

To construct 35S:ERF012 transgenic plants, the open reading frame (ORF) sequences of ERF012 were amplified by polymerase chain reaction (PCR) using specific primers (Supplemental Table S1). The ERF012 CRISPR vector was generated according to the method of Yang [52 (link)]. To construct the pERF012:GUS vector, the promoter sequence of ERF012 was amplified by PCR using the specific primers, then fused with the Sma I restriction endonuclease-digested DX2181 using a 5× infusion kit (Clontech, Takara, Beijing, China). The Agrobacterium GV3101-mediated flower-dip method was used to generate the transgenic lines [53 (link)]. For the transcription activation assay of ERF012, the effector vectors were constructed according to the methods [45 (link),54 (link)]. The ERF012 CDS sequence fragment was amplified by PCR, then inserted into the binary vector GAL4DB [55 (link)] using the EcoR I restriction endonuclease. For the yeast one-hybrid assay, the promoter of 4CL and C4H were inserted into a pHis2 vector using Sma I by the 5× infusion kit (Clontech, Takara, Beijing, China), and the CDS sequences of ERF012 were inserted into the pGADT7-rec2 vector using Xma I and Xho I with specific primers. All the specific primers are listed in Supplementary Table S1.

Genes for HLA-A*0201-resticted, AURKA_207-215-specific TCR-α and TCR-β chains [11 (link)] were cloned with an InFusion kit (Takara Bio) into a pSplice-a2Ab-siTCR vector [33 (link)] to generate ‘siAUK’ vectors (S1 Fig). The constant regions of the α- and β-chains were codon optimised to escape interference by the siTCR expressed by the pSplice-a2Ab-siTCR vector. An existing vector bearing the anti-AURKA α- and β-chains (Takara Bio) [11 (link)] was used as a control (‘coAUK’).
Ecotropic, VSV-G pseudotyped retrovectors were generated by transient co-transfection of HEK-293T cells by Calcium-Phosphate precipitation with a Retrovirus Packaging Kit (Takara Bio). This supernatant was filtered, and used to transfect PG13 packaging cells to generate GaLV-pseudotyped retrovirus particles, as described previously [4 (link)].

The cD-0 was subjected to site-directed mutagenesis by overlap extension PCR (OE-PCR) and In-Fusion® assembly for constructing 17 mutated SVA cDNA clones, cD-1 to -17. The cD-1 to -7 harbored seven types of mutated KLFMs; the cD-8 to -17 possessed 10 types of mutated PFMs. Briefly, the cD-0 was subjected to PCR separately using two pairs of primers (Table 1), forward primer 1 (FP1)/reverse primer 1 (RP1) and FP2/RP2. The first step included two independent PCRs to amplify fragment I and II from the cD-0 using the FP1/RP1 and FP2/RP2, respectively. Fragment I and II were used as templates in a tube for the second step of PCR using the FP1/RP2. The fragment I and II were fused into a longer one, followed by homologous recombination with BamH I/Pme I-digested cD-0 using the In-Fusion® Kit (Takara, Dalian, China) to construct a mutated cDNA clone. Seventeen mutants were subjected to Sanger sequencing, purification, and agarose gel electrophoresis for confirming their mutated sites.

The P. waltl Cldn6 sequence was retrieved from the P. waltl genome. Amino acids at positions 140–157 were removed to create the PwCldn6Δ sequence, where the ECL2 domain was removed. The T2A-H2B-EBFP2 sequence was inserted to the 5′ end of the stop codon in wild-type P. waltl Cldn6 and the PwCldn6Δ sequence, as a selection marker. The recombinant sequences were synthesized as double-stranded DNA fragments (IDT gblock) and inserted into a piggyBac-CAG expression plasmid using an infusion kit (Takara). Plasmids were transformed into One Shot Stbl3 chemically competent Escherichia coli (Thermo Fisher, C737303).

Construction of LV::S_WA1 and LV::S_WA1-_ΔF2P was described elsewhere [14 (link),15 (link)]. Briefly, codon-optimized S sequences (1-1262) from the ancestral S_WA1 and Beta SARS-CoV-2 strains were amplified and inserted into the pFlap lentiviral plasmid by restriction/ligation between BamHI and XhoI sites, between the native human ieCMV promoter and a mutated Woodchuck Posttranscriptional Regulatory Element (WPRE) sequence. A directed mutagenesis was performed by use of a Takara In-Fusion kit to introduce the 2 proline mutations in S_WA1 or S_Beta, on the corresponding pFlap plasmids.

For complementation of the cnx2-2 phenotype, a genomic fragment including the promoter region from position −1224 and UTRs was amplified by PCR using the primers A1F and A1R and Phusion polymerase as per the manufacturer's instructions (New England Biolabs). The PCR fragment was cloned in a pUC-derived cloning plasmid using HindIII and KpnI to cut the vector and an In-Fusion Kit (Takara) to insert the fragment. After confirming the sequence, the gene fragment was cut out using AscI and PacI and ligated into a pBIN-derived binary vector containing a hygromycin resistance marker. cnx2-2 plants were transformed using the floral-dip method and Agrobacterium tumefaciens (strain GV3101). Successfully transformed plants were selected on medium containing 25 µg ml⁻¹ hygromycin B.