Figure 2
From: A novel approach to the generation of seamless constructs for plant transformation
Overview of SRL used with In-Fusion™ cloning. Red letters indicate recognition sites and black triangles cleaving sites of LguI and BaeI. Red arrows indicate where In-Fusion™ enzyme exonuclease activity will occur. A. Spacer-removal linearization (SRL) of pAUrumII (to pAUrumIILIN) with type IIS RE LguI. The spacer holds the LacZα coding sequence with its promoter. B. SRL of pAUrumIII (to pAUrumIIILIN) with type IIB RE BaeI. C. Extended primers used in PCR amplification of gfp. The In-Fusion™-ready product, gfpIFR, has 15 nt of homology with the 35S promoter 3’-end and 15 nt of homology with the NOS terminator 5’-end. Moreover, it has the monocot Kozak consensus sequence AACC in front of the start codon. D. In-Fusion™ reaction and assembly of pAUrumIILIN or pAUrumIIILIN with gfpIFR and subsequent transformation of non-ligated construct to E. coli, where repair and ligation will occur. There are no unwanted nucleotides in the final construct.