Process for the manipulation of nucleic acids

A method for removing at least two portions of insert nucleic acid from a genomic polynucleotide in a host cell, said method comprising the steps of:

• a) preparing the genomic polynucleotide comprising at least a first and a second insert nucleic acids, wherein i) the first insert nucleic acid is flanked by first recombination sites in the same orientation which are identical to each other and have a first nucleic acid sequence ii) the second insert nucleic acid is flanked by second recombination sites in the same orientation which are identical to each other and have a second nucleic acid sequence which shares no more than 98% sequence identity with the first nucleic acid sequence and iii) any further recombination sites have a nucleic acid sequence that shares no more than 98% sequence identity with the first or second nucleic acid sequences;
• and
• b) exposing the genomic polynucleotide to a recombinase that recognises the first and second recombination sites such that the identical recombination sites recombine resulting in the excision of the insert nucleic acid flanked by identical recombination sites but without the removal of genomic polynucleotide sequence which is not flanked by identical recombination sites.

A host cell comprising a host cell genome polynucleotide containing a first recombinantly engineered region and a second recombinantly engineered region, wherein a first recombination site scar is adjacent to the first recombinantly engineered region and a second recombination site scar is adjacent to the second recombinantly engineered region;

• wherein the first and second recombination site scars have different polynucleotide sequences which are less than 98% identical to each other and optionally less than 98% identical to the polynucleotide sequence of any further recombination site scar present in the host cell genome polynucleotide.

A prokaryotic genomic polynucleotide or a eukaryotic chromosome comprising at least two recombination site scars adjacent to at least two recombinantly engineered regions, wherein each recombination site scar has a different polynucleotide sequence.

An engineered host cell comprising single copies of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 recombination sites in the host cell genomic polynucleotide, wherein each recombination site has a nucleotide sequence which is less than 98% identical to the other recombinations sites.