Pcmv6 an myc ddk

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.

pCMV6-AN-Myc-DDK (PS100016; RRID:SCR_021264) and pCMV6-AN-mRFP (PS100049; RRID: SCR_021265) were purchased from Origene. Inducible GFP-RNAseH1 plasmid was generated via restriction cloning an RNH1-GFP cassette from pEGFP‐RNASEH1, a gift from Andrew Jackson and Martin Reijns (RRID: Addgene_108699) using BglII + NotI and cloning into the BamH1 NotI sites of pENTR1A (RRID:SCR_021263). The RNH1-GFP cassette was then transferred to the pCLX-pTF-R1-DEST-R2-EBR65 lentiviral plasmid, a gift from Patrick Salmon (RRID:Addgene_45952) via an LR (recombination reaction between attL and attR sites) recombination reaction, creating a single vector that expresses the Tet-repressor and blasticidin resistance gene under the control of a constitutive promoter and containing the RNHA1-GFP cassette under the control of a Tet-repressor regulated promoter. SF3B1-K700E and RNAseH1 D210N mutations were created by site-directed mutagenesis.

Plasmid construct were ordered at Genscript. DNA sequences for ACKR2-WT and ACKR2-V41A were inserted into vector pCMV6-AN-myc-DDK (ORIGENE, Cat #PS100016). Transformation protocols were followed as recommended by the manufacturer. Briefly, plasmids were transformed into DH5a cells, amplified, and purified using the ZymoPURE II Plasmid Maxiprep kit (Cat #D4203). Plasmids were transfected into Chinese Hamster Ovary (CHO-k1 ATCC® CCL-61) cells using lipofectamine (ThermoFisher, Cat #15338100) and manufacture recommendations were followed. Properly transfected cells were selected using the antibiotic G418 sulfate (ThermoFisher, Cat #10131035). Transfected cells were grown in F12 media (10 μg/ml penicillin, 10 μg/ml streptomycin; Gibco Cat #21127-022) with 10% FBS (HYCLone Cat #SH30071.01). Cell media was changed every 48 to 72 hours depending on cell confluency levels.