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. Author manuscript; available in PMC: 2014 Aug 25.
Published in final edited form as: Nat Biotechnol. 2013 Jan;31(1):76–81. doi: 10.1038/nbt.2460

Figure 1. The LIC assembly approach to generating TALEN genes.

Figure 1

(a) Architecture of the previously published Δ152/+63-AvrBs3-like TALEN that harbors a truncated N terminus (Δ152), an invariant recognition domain for thymine followed by a stretch of several repeat units, and a truncated and optimized C terminus (+63) fused to a FokI domain. One repeat unit is highlighted indicating the 34 amino acids and the RVDs in position 12 and 13 that were used. At right, a model of a minimal cloning unit consisting of two consecutive repeat units flanked by ID1 and ID2. (b) The library of the 64 possible 2-mer combinations with ID 1/2, 2/3, 3/4 and 4/1. (c,d) First level assembly: three 2-mers containing the target sequences are picked with alternating ID combinations (c); three 2-mers are assembled to generate a complex of six tandem repeats (6-mer) into a level 1 backbone that encodes for kanamycin resistance (kanaR) (d). (e,f) Second level assembly: assembly of a full TAL effector gene is achieved upon combining three 6-mers (e), and a mammalian expression vector containing the Δ152/+63-AvrBs3-like TAL effector backbone with a C-terminal FokI nuclease domain (f). Additional features of the mammalian expression vector (level 2 vector) are highlighted (pCMV, cytomegalovirus-promoter, ampR, bacterial resistance gene for ampicillin; NLS, nuclear localization signal; ATG, start codon; STOP, stop codon; T7, promoter sequence for T7 RNA polymerase; XhoI, XbaI, NotI, recognition sequences of the restriction enzymes XhoI, XbaI and NotI, respectively). To prevent undigested fragments from being propagated to the next level of assembly, we switched antibiotic resistance at each level.