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Figure 1 | Plant Methods

Figure 1

From: UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Figure 1

Overview of the USER™ reaction. A. The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI, which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B. The two USER™ cassettes designed and used in this study, the USERTC-TG and the USERTC-CC.

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