The Gateway® reactions are in vitro versions of the
integration and excision reactions. Your goal is to move your
gene (or genes) from one vector backbone to another.
Let's first consider the LR Reaction for making expression
1. Your sequence of interest (X) is cloned
in an Entry vector that is transcriptionally silent, Kmr, and
is flanked by two recombination sites (attL1 and attL2).
2. You want to move this sequence of interest to a Destination
vector. It contains all the sequence information required for
expression, and is Apr. This plasmid also contains two recombination
sites (attR1 and attR2) that flank a gene for negative
3. You combine the two plasmid DNAs, each with sequences flanked
by recombination sites that do not recombine with each other,
but will recombine with sites resident on the other molecule.
4. LR Clonase is comprised of Int, IHF, and Xis; you are
doing an in vitro version of the excision reaction.
5. Att1 and att2 sites confer directionality and specificity
for recombination, so that only attL1 will react with
attR1, and attL2 with attR2.
6. Two recombination events occur to make the expression clone,
one between attL1 and attR1 and the other between
attL2 and attR2.
7. The product of these two recombination events is the plasmid
construct that you want and a by-product (labeled as the donor
vector). The desired plasmid is under two forms of selection:
antibiotic resistance and negative selection. Selecting for Apr
eliminates the starting vector and the by-product; the presence
of the negative selection marker eliminates the destination vector
and co-integrate molecules.