Figure 3.

(A) Destination Vector pDEST17, for expressing His6 fusions in Escherichia coli. (B) Colonies resulting from transformation of DH5α cells with 2 μL (of 22 μL) of LR reactions. The negative control contained all components except the Entry Clone DNA. pDEST17 was linearized at the unique NcoI site prior to mixing with Entry Clone DNA. (C) The Expression (attB) clone pEXP17-eIF4e, which resulted from the LR reaction with pENTR203-eIF4e and pDEST17. The subcloned eIF4e gene has replaced the chloramphenicol resistance-ccdB segment, with the amino end of the gene downstream of the T7 promoter and the His6 tag in frame with the eIF4e open reading frame. The recombination reactions convert the attR sites to attB sites (25 bp). (D) Miniprep DNA of single colonies (Expression Clones) from each reaction. Lane 1, eIF4e; lane 2, tyrosine kinase; lane 3, transferrin receptor; lane 4, β-adaptin; lane 5, MAP4; lane 6, Gus; lane 7; TetR. M, supercoiled DNA ladder.