Construction of SABRs

Single molecules encoding for β2-microglobulin and HLA were synthesized as gBlocks (IDT) and amplified using primers SS-Fwd and CD28-Overlap-HLA-Rev. CD3ζ/CD28 signaling domains were cloned from the J3 CAR (A gift from Pin Wang) using primers CD28Intracell-Fwd and XhoI-CD3z-Rev. The two parts of SABRs were assembled via PCR or via InFusion HD cloning kit (Takara). A synthetic 2kb fragment of non-specific stuffer DNA (IDT) flanked by BsmBI sites was cloned in place of the epitope. To clone a given epitope into a SABR vector, the vector was first linearized by BsmBI digestion (NEB) and gel purified using Nucleospin Gel and PCR kit (Takara). A single stranded oligonucleotide containing overlaps with the vector and the epitope was synthesized (IDT). The oligonucleotides were amplified using KOD polymerase (Milipore) and Oligo-Insert-Fwd and Oligo-Insert-Rev. Amplified oligonucleotide was cloned into the linearized SABR vector using InFusion HD cloning kit (Takara). All cloning reactions were transformed into Stellar competent cells (Takara), grown on LB+Agar plates containing 100 μg/ml Carbenicillin (Life Technologies), and individual colonies were inoculated in liquid culture. Plasmid minipreps were performed using Zyppy Miniprep kit (Zymo). Plasmids were verified by Sanger sequencing (Laragen).