organism: synthetic construct
description: Sequences from Hubbard, et al., 2015. The specificity profiles of PACE evolved TALENs targeting ATM target sites using an in vitro selection method. Briefly, preselection libraries of > 10^12 DNA sequences, each theoretically containing at least 10 copies of all possible DNA sequences with six or fewer mutations relative to the on-target sequence (TGAATTGGGATGCTGTTT-random spacer- TTTATTTTACTGTCTTTA), were digested with in vitro-translated TALEN. Cleaved library members harbored a free 5 monophosphate that enabled them to be captured by adaptor ligation. We isolated DNA fragments of length corresponding to 1.5 target sites (an intact target site and a repeated half-site up to the point of TALEN-induced DNA cleavage) by gel purification. High-throughput sequencing of TALEN-treated or control samples that survived this selection revealed the abundance of all TALEN-cleaved sequences as well as the abundance of the corresponding sequences before selection. In the control sample, all members of the preselection library were cleaved by a restriction endonuclease (NEB Acc65I) at a constant sequence to enable their capture by adaptor ligation and isolation by gel purification. Selections were indexed with the first 5 bp of sequence. Thus, the first 5bp of each sequences identifies which selection condition the sequence originated from: For sequences fastq files:"ACTGT" = Canonical 20 nM, "CTGAA" = Canonical 10 nM, "TGACT" = Canonical 5 nM, "TGCAA" = Canonical 2.5 nM, "GCATT" = L3-1 20 nM, "CATGA" = L3-1 10 nM, "ATGCT" = L3-1 5 nM, "CTAGT" = L3-1 2.5 nM, "GCTAA" = L3-2 10 nM, "CAGTT" = L1-1 10 nM. Pre-selection library sequences are in of the format NNNNGGTACCCCACTCCGCGT