Patent ID: 7935862

Claim:
A method for targeted nucleotide sequence stacking in a genome of a host cell, the method comprising the steps of: (a) providing a host cell having a genome with a target sequence comprising: (i) a first heterologous sequence of interest; (ii) a truncated functional sequence; (iii) a host homology sequence located between the first heterologous sequence of interest and the truncated functional sequence; and (iv) a target recombinase recognition site located between the first heterologous sequence of interest and the host homology sequence; (b) introducing into the host cell a donor sequence comprising: (i) a second heterologous sequence of interest or fragment thereof homologous to the first heterologous sequence of interest; (ii) a donor homology sequence homologous to the host homology sequence; (iii) a sequence that completes the truncated functional sequence, which is located between the second heterologous sequence of interest or fragment thereof and the donor homology sequence; (iv) a first donor recombinase recognition site located between the donor homology sequence and the sequence that completes the truncated functional sequence; (v) a second donor recombinase recognition site located between the sequence that completes the truncated functional sequence and the second heterologous sequence of interest or fragment thereof; and further wherein the first and second donor recombinase recognition sites are oriented relative to one another such that the sequence that completes the truncated functional sequence is excisable in the presence of a recombinase; and (vi) a third heterologous sequence of interest located between the second heterologous sequence of interest and the second donor recombinase recognition site; (c) obtaining in the host cell a first recombination product comprising: (i) a first recombined sequence resulting from homologous recombination of the first heterologous sequence of interest and the second heterologous sequence of interest; (ii) a second recombined sequence resulting from homologous recombination of the host homology sequence and the donor homology sequence; (iii) a restored functional sequence comprising the sequence that completes the truncated functional sequence, the truncated functional sequence, the second recombined sequence located between the sequence that completes the truncated functional sequence and the truncated functional sequence, and the first donor recombinase recognition site located between the sequence that completes the truncated functional sequence and the the second recombined sequence; (iv) the third heterologous sequence of interest located between the first recombined sequence and the restored functional sequence; and (v) the second donor recombinase recognition site located between the third heterologous sequence of interest and the sequence that completes the restored functional sequence.