Tal-mediated transfer dna insertion

A method for the targeted insertion of exogenous DNA into a plant comprising the steps of (i) transforming isolated plant cells with

• (A) a first binary vector comprising a promoter-less cassette comprising (a) a right border sequence linked to (b) a partial sequence of the Ubi7 intron 5′-untranslated region;
• (c) an Ubi7 monomer-encoding sequence fused to a mutated acetolactate synthase (ALS) gene;
• (d) a desired nucleotide sequence;
• and (e) a terminator sequence, wherein the desired nucleotide sequence is not operably linked to a promoter;
• and
• (B) a second binary vector comprising (a) a right border;
• (b) a forward expression cassette and a reverse expression cassette, each comprising a modified TAL effector operably linked to a strong constitutive promoter, and a terminator sequence;
• and (c) a sequence encoding isopentenyl transferase (ipt), wherein the modified TAL effector is designed to bind the desired nucleotide sequence within an intron of potato'subiquitin-7 (Ubi7) gene;
• and (ii) culturing the isolated plant cells under conditions that promote growth of plants that express the desired nucleotide sequence;
• wherein no vector backbone DNA is permanently inserted into the plant genome.

A DNA construct comprising a promoter-less cassette comprising (a) a right border sequence linked to (b) a partial Ubi7 5′-untranslated intron sequence;

• (c) an Ubi7 monomer-encoding sequence fused to a mutated acetolactate synthase (ALS) gene;
• (d) a desired nucleotide sequence;
• (e) a terminator sequence;
• and (f) a left border, wherein the desired nucleotide sequence is not operably linked to a promoter.

A kit for targeted insertion of exogenous DNA into a plant comprising:

• (A) a first binary vector comprising a promoter-less cassette comprising (a) a right border sequence linked to (b) a partial sequence of the Ubi7 intron 5′-untranslated region;
• (c) an Ubi7 monomer-encoding sequence fused to a mutated acetolactate synthase (ALS) gene;
• (d) a desired nucleotide sequence;
• and (e) a terminator sequence, wherein the desired nucleotide sequence is not operably linked to a promoter;
• and
• (B) a second binary vector comprising (a) a right border;
• (b) a forward expression cassette and a reverse expression cassette, each comprising a modified TAL effector operably linked to a strong constitutive promoter, and a terminator sequence;
• and (c) a sequence encoding isopentenyl transferase (ipt), wherein the modified TAL effector is designed to bind the desired nucleotide sequence within an intron of potato'subiquitin-7 (Ubi7) gene.