Pmxs ires blasticidin

Wild-type cDNAs (TRMT61B, clone BC010365.1; MTU1 (TRMU), clone CU013043; MTO1, clone BC011051) were obtained from the ORFeome or Mammalian Genome Collection. These cDNAs were in a Gateway entry vector (pENTR221 or pCMV-SPORT6) or cloned into pDONR201 using PCR with KAPA HiFi followed by recombination. The TRMT61B cDNA was missing a stop codon, which was added by PCR using the primers BB-477 (5′-aaaaagcaggctaccATGCTAATGGCATGGTGCCG-3′) and BB-478 (5′-agaaagctgggtttattaGTTAAGTTGTGGTTTGACC-3′) with KAPA HiFi followed amplification with attB1 and attB2 adapters then by gateway cloning into pDONR201. All full-length cDNAs were recombined into Gateway converted pMXs-IRES-Blasticidin with LR Clonase II (Thermo Fisher). All cDNAs were thoroughly sequenced by Sanger sequencing for verification initially and after all PCR manipulations.

Wild-type cDNAs (AFG3L2, clone BC065016; COX10, clone BC000060) were obtained from the ORFeome or Mammalian Genome Collection. These cDNAs were in a Gateway entry vector (pENTR221 or pCMV-SPORT6) or cloned into pDONR201 using PCR with KAPA HiFi followed by recombination. All full-length cDNAs were recombined into Gateway-converted pBABE-puro and pMXs-IRES-Blasticidin with LR Clonase II (Thermo Fisher Scientific). All cDNAs were thoroughly sequenced by Sanger sequencing for verification initially and after all PCR manipulations. Site-directed mutagenesis of E575Q in AFG3L2 was performed using Agilent primer design with PCR (KAPA HiFi), purification, DpnI (NEB) digest, and transformation into XL1 Blue chemically competent cells. Sanger sequencing was used to validate mutagenesis. Mutant cDNA was recombined into Gateway-converted pBABE-puro and pMXs-IRES-Blasticidin with LR Clonase II.