Fig. 1. Schematic overview of the TALEN assembly process (according to [33]) and the resulting plasmids. Six monomers are assembled into hexamers corresponding to the desired target site in a golden-gate type reaction from a library consisting of 72 monomers (18 positions of the TALEN target site and 4 different RVDs), allowing assembly of multiple fragments in the desired order in a single step. Three of these hexamers are then ligated in a second golden gate-type reaction into the plasmid containing the first RVD (always NG) and the last half RVD (HD, NG, NI or NN, depending on the target site). Correct insertion of the target sequence needs to be verified by colony PCR (insert size: 2.2 kbp). Clones indicated to be positive by colony PCR should be verified by restriction digest with AfeI (expected fragment lengths: 5 kbp, 2.2 kbp and 165 bp; exemplary shown in Fig. S1) as well as sequencing of the inserted fragment. fcpA: FcpA ( = Lhcf1) promoter; FokI: endonuclease; N-/C-term: N and C terminus, respectively; Nat: nourseothricin resistance cassette; NR: nitrate reductase promoter; RVD: repeat variable di-residue; Sh ble: Zeocin resistance cassette.
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