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Generation of pDEST-MyGene plasmids　Outline of promoter fusion gene construction

A protocol for generation of pENTR plasmids by attB-attached PCR and BP recombination (in preparation)

There are two major strategies to make a Gateway plasmid that carry a promoter fragment.

Strategy 1) A promoter fragment either cut out by restriction enzymes or amplified by PCR is inserted by ligation between attL1 and attL2 sites of Gateway vectors such as pENTR1A (Invitrogen)

Strategy 2) A promoter fragment is amplified using a pair of primers with additional attB1and attB2 sequences, respectively, at their 5' ends. Amplified DNA fragment is inserted into Gateway vectors such as pDONR201 (Invitrogen) by site-specific recombination using BP Clonase.