Plasmid Construction and Characterization

pcDNA3.1(+)-THRAP3-FLAG, pcDNA3.1 (+)-THRAP3-ΔN190-FLAG, pcDNA3.1 (+)-THRAP3-ΔC359-FLAG and pcDNA3.1 (+)-THRAP3-ΔNC-FLAG plasmids were kindly provided by professor Woan-Yuh Tarn, National Taiwan University, Taipei, Taiwan (20 (link)). The pRFP2GFP-ATM construct was generated using the pmaxGFP vector as a backbone. The RFP (red fluorescent protein) sequence was amplified by polymerase chain reaction (PCR) from pmTagRFP-T2-N1 (Michael Davidson Lab via Addgene) using the following primer sequences: Forward 5′-ATGGTGTCTAAGGGCGAAGAG-3′ and Reverse 5′-GCGGTACCGTCGACTGCA-3′ plus 20 bp of homology and HindIII and SalI sites in the reverse primer. This PCR product was inserted into the pmaxGFP vector upstream of the turbo-GFP (green fluorescent protein) open reading frame (ORF) between the KpnI and AgeI sites using Gibson assembly (NEB). The sequence encoding the ATM exon20-intron20-exon21 was PCR amplified from 293T derived genomic DNA using the following primer sequences: Forward 5′- GTGAAGCTTTCCAATGTGTGTTCTTTGTATC-3′ and Reverse 5′- TACGTCGACCTCAAGCAAAGTTTTAAG-3′, and cloned into the pRFP2GFP vector’s ORF between RFP and GFP tags using novel HindIII and SalI sites. pCMV6-AN-Myc-DDK was purchased from Origene (PS100016). The ORF of THRAP3 was inserted in this plasmid using SgfI and MluI sites. THRAP3, cloned in this vector, was expressed as the N-terminal Myc-DDK-tagged protein.