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Pools of Arabidopsis RNA were isolated and used as template to create pools of cDNA's. These pools were then linkered and ligated directionaly to GFP, creating fusions where GFP resides at the N-terminus of the expressed protein. Fusion junctions were created both within open reading frames and upstream of initiating ATG codons. 1 in 3 fusions are expected to be in frame with the native coding sequence. GFP is carried in a purpose built plant expression vector, pEGAD. pEGAD places a Ala10 flexible linker between GFP and the cloned insert. Expression in planta is driven by the CamV 35S promoter, and selection is conferred by resistence to the herbicide Basta. For more details about pEGAD, see the section on vectors and maps. |
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