Pbad hisc

The E. coli strain AF1000 (MC4100, relA1⁺) [18 (link)] was used for expression of the genes rx, rxHis₆, t3 and zwf, as well as for production of 3HB. The E. coli strain DH5α was used for all cloning procedures. The plasmid pJBGT3RX was used for 3HB production. It was constructed by SLIC insertion of rx and t3 to pKM1D, a pACYC184-derived low copy number plasmid with ori p15A, a lacUV5 promoter, a multiple cloning site, the lacIq repressor and a chloramphenicol resistance gene (Additional file 1: Figure S1). The plasmid pBADzwf for increasing intracellular NADPH concentrations was constructed from pBAD/HisC (Invitrogen), a plasmid with the pBR322 ori, the araBAD promoter and an ampicillin resistance gene. From DH5α, zwf was amplified by PCR with complementary tails to the NcoI and EcoRI sites of pBAD/HisC. The plasmid was digested with EcoRI and NcoI followed by SLIC assembly [19 (link), 20 ] to zwf. The plasmid pTrc99RXHis₆ for purifying rxHis₆ was constructed from pTrc99A [21 (link)], a plasmid with pBR322 ori, the lacI repressor, the trc promoter and an ampicillin resistance gene. From pJBGT3RX, the gene rx was amplified by PCR with complementary tails to the NcoI site of pTrc99A. The plasmid was digested with NcoI followed by SLIC assembly [20 ] to rx.

The PRDM9^B allele was purchased from OriGene (Rockville, MD). Oligonucleotide primers were designed to include a 5’ V5 epitope tag and used to amplify the full-length PRDM9 and cloned into pCEP4 expression vector (Invitrogen) to create pCB09. A 6X-HIS-3X-FLAG tag was inserted in frame replacing the V5-tag using yeast-based homologous recombination [53 (link)]. The zinc-finger arrays for both PRDM9^A and PRDM9^C were amplified from human genomic DNA [7 (link)] and cloned into pBAD-HisC (Invitrogen). These zinc-finger arrays were subcloned into the pCEP4 vectors using restriction enzymes AflII and HindIII (NEB) to create full-length tagged versions of FLAG-PRDM9^C (pCB51), V5-PRDM9^C (pCB47), and FLAG-PRDM9^A (pCB53), and V5-PRDM9^A (pCB48) for expression in mammalian cell culture. The V5-PRDM9^C-G278A allele was created using QuikChange II site-directed mutagenesis (Agilent Technologies) to change glycine 278 to alanine to create pCB56. All cloning oligonucleotides are listed in S3 Table.