Screening vector with T7 promoter, adds N-terminal His tag, TEV, LICs, GFP; amp resistance; ligation independent cloning (LIC).
Comments:
Standard LIC cloning (linearize the vector with SspI, then treat insert and vector with T4 polymerase), using these sequences for the PCR primers:
Forward: 5'-TACTTCCAATCCAATGCC-3' + GOI sequence
Reverse: 5'-TAATCCACTTCCAATAGC-3' +GOI sequence
These primers leave an alanine scar on both the N and C terminus of the GOI resulting in the following protein sequence:
His6-TEV(cleavage site)-Alanine-GOI-Alanine-GFP
Publications:
None
Authors:
M.Donnelly
Midwest Center for Structural Genomics
PSI
Midwest Center for Structural Genomics