Midi prepped

We introduced point mutations into DdCBE expression plasmids through site-directed mutagenesis. In brief, we amplified plasmids with mutagenesis primers before mutagenesis with the Q5 Site-Directed Mutagenesis Kit (NEB), and the results were confirmed with Sanger sequencing. For assembly of plasmids encoding interface mutant DdCBEs, the mini-prepped mutant expression plasmid was added to a solution containing module vectors (each encoding a TALE array^{22 (link)}), BsaI-HFv2 (10 U), T4 DNA ligase (200 U) and buffer in a single tube. For the TALE-free construct, instead of module vectors we used oligonucleotides (Macrogen) (forward, 5′-CTGAGTGGTAGTGGTAGTGGTTCTGG-3′; reverse, 5′-ACCGCCAGAACCACTACCACTACCAC-3′) that encode a flexible linker N-(SG)₃-C. amino acid sequences of the TALE arrays for each target are indicated in Supplementary Fig. 16. The restriction and ligation reactions were performed in a thermocycler, with 20 cycles at 37 °C and 16 °C for 5 min each, followed by final incubations at 50 °C for 15 min and 80 °C for 5 min. Ligated plasmids were chemically transformed into Escherichia coli DH5ɑ; plasmids from the transformants were subjected to Sanger sequencing to analyze the identity of the constructs. Final plasmids were midi-prepped (Qiagen) for cell transfection.

The sequences of 5cc7α, 5cc7βG, CD4 WT, and CD4 T1 constructs were previously described (Lee et al., 2022 (link)). Full-length CD3δ, -ε, -γ, and -ζ were encoded on a poly-cistronic construct as previously described (Holst et al., 2008 (link); Kuhns and Davis, 2007 (link)).
The following CD4-T1 constructs: T1-TMD, T1-Palm (2C), T1-Palm (3C), T1-TP (2C), and T1-TP(3C) were cloned by conventional molecular biology techniques, including PCR-based mutagenesis where needed, into pUC18 via 5’ EcoRI and 3’ HindIII. After sequence verification they were subcloned into pP2 puromycin resistance MSCV vector (MCS-IRES-puro) via 5’ XhoI and 3’ BglII, midi-prepped (QIAGEN), and sequence-verified. Mutated codons are shown in bold uppercase letters, while the motif under interrogation is shown in bold and underlined (any non-mutated codons within the motif are bold lowercase letters and underlined). The sequences for these constructs are as follows: