The corresponding Atg8ΔR variant was constructed by site-directed mutagenesis using the follow primers: Forward, ACA TTT GGC TAG CAG TCT TTT ATA TG, Reverse, AGA CTG CTA GCC AAA TGT ATT TTC TC.
A FLAG-tagged HOT1 homeobox deletion variant was constructed by PCR amplification of the pcDNA3.1-FLAG vector using primers with site-specific overhangs (Table III; Metabion) following the QuickChange II Site-directed mutagenesis kit protocol (Stratagene; Zheng et al, 2004).
The mammalian expression vectors for Flp variants were constructed by cloning the genes for the variants into pOG44 (Invitrogen) in place of the FLP-F70L variant gene.
A library of enzyme variants was constructed by staggered extension process (StEp) using the genes that code for the l-ASNases from Erwinia chrysanthemy (ErL-ASNase) and Erwinia carotovora (Ecal-ASNase) and screened using activity assays.
Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry.
The HsmtSSB deletion variants were constructed by cloning of PCR fragments containing the coding region for HsmtSSBΔN, ΔC and ΔNΔC into the NdeI site of the pET11a vector.
Both Y137A and Y137F NAL variants were constructed by site-directed mutagenesis and expressed in E. coli as for the wild-type enzyme.
An additional control set of background variants was constructed by randomly sampling the 1000 Genomes EUR dataset for SNPs that were matched for distance to the nearest TSS.
All newly described Vibrio 5S rRNA variants were constructed by in vitro mutagenesis using the manufacturer's protocol provided with the Sculptor™mutagenesis kit.
A panel of PAV variants was constructed by replacing the cleavage motif of Lock1 with previously identified sequences having demonstrated susceptibility to different MMPs (Table 1 ).
