# FLP-FRT recombination
FLP-FRT recombination is a site-specific recombination system in which the FLP (flippase) recombinase, encoded by the 2 microns plasmid of the budding yeast *Saccharomyces cerevisiae*, catalyzes DNA rearrangements at short recognition sequences called FRT (FLP recognition target) sites. In its native host it drives plasmid copy-number amplification; in biotechnology it is used to integrate, excise, invert, or exchange defined DNA segments in yeast, mammalian cells, plants, and mice.
| Key fact | Detail |
|---|---|
| Origin | FLP is encoded by the 2 microns plasmid of *S. cerevisiae* and catalyzes recombination between two repeated segments on the plasmid[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC367081/) |
| Native FRT location | The FRT sites lie within two 599 bp inverted repeats of the 2 microns plasmid[2](https://pubmed.ncbi.nlm.nih.gov/7932750/) |
| Minimal FRT site | Two inverted 13 bp FLP binding sites surrounding an 8 bp core (spacer) region; the engineered site used in applications is 34 bp[2](https://pubmed.ncbi.nlm.nih.gov/7932750/) • [3](https://pmc.ncbi.nlm.nih.gov/articles/PMC1764711/) |
| Mechanism | Conservative strand exchange through a Holliday junction intermediate, in two temporally separated cleavage-exchange steps[2](https://pubmed.ncbi.nlm.nih.gov/7932750/) • [4](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014) |
| Temperature optimum | FLP activity peaks at 30 °C and drops sharply at 37 °C, the mammalian body temperature; the evolved variant FLPe works more efficiently in mammalian cells[3](https://pmc.ncbi.nlm.nih.gov/articles/PMC1764711/) • [5](https://www.jax.org/news-and-insights/jax-blog/2014/November/going-beyond-cre-lox) |
| Outcome depends on site orientation | Inverted (head-to-head) FRT sites give inversion of the intervening DNA; direct (head-to-tail) sites give deletion[4](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014) |
| Relation to Cre-lox | A parallel system from bacteriophage P1; FLP-FRT is generally less efficient in mammals and is often used alongside Cre-lox for independent manipulations[5](https://www.jax.org/news-and-insights/jax-blog/2014/November/going-beyond-cre-lox) • [6](https://digitalcollections.ohsu.edu/record/42751/files/Jahncke.Jennifer.2024.pdf) |
## Biological role in the 2 microns plasmid
The 2 microns plasmid is a small multi-copy DNA element that resides in the yeast nucleus at 40 to 60 copies per haploid cell[7](https://www.sciencedirect.com/science/article/pii/S0147619X13000292). Its FLP gene encodes a conservative site-specific recombinase, a member of the integrase family of recombinases[2](https://pubmed.ncbi.nlm.nih.gov/7932750/). The key components of the plasmid's amplification system are the Flp recombinase and a pair of inverted FRT sites whose positions relative to the plasmid replication origin are strongly asymmetric[7](https://www.sciencedirect.com/science/article/pii/S0147619X13000292). Recombination between these inverted repeats inverts a segment of the plasmid[8](https://pubmed.ncbi.nlm.nih.gov/2648010/), and the flip-flop of this segment underlies the copy-number amplification mechanism[7](https://www.sciencedirect.com/science/article/pii/S0147619X13000292).
## The FRT site and the recombination mechanism
The minimal FRT target contains two inverted 13 bp FLP binding elements that flank an 8 bp core region, where strand exchange occurs[2](https://pubmed.ncbi.nlm.nih.gov/7932750/). FLP nicks the top and bottom strands of the FRT site at the margins of the core, and these nicks are the sites of strand exchange; recombination therefore generates heteroduplex DNA in the core region. Sequence differences between the cores of two FRT sites inhibit their recombination, an important property for engineering orthogonal sites[2](https://pubmed.ncbi.nlm.nih.gov/7932750/).
**Strand exchange proceeds in two steps.** Like other tyrosine recombinases, FLP cleaves DNA with an active-site tyrosine nucleophile, forming a 3′-phosphotyrosyl intermediate and a 5′-hydroxyl group; the 5′-hydroxyl then attacks the phosphotyrosyl bond on the partner DNA to reseal the strands in the recombinant configuration. The first cleavage-exchange step yields a Holliday junction intermediate, which isomerizes and is resolved by a second exchange into reciprocal recombinants[4](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014).
**The active site is shared between monomers.** FLP is unusual in that it assembles its active site at the interface of two neighboring monomers: the tyrosine nucleophile Tyr-343 is donated in trans by one monomer into the proactive site of the other. The catalytic residues are Arg-191, Asp-194, Lys-223, His-305, Arg-308, and Trp-330, with Tyr-343 providing the cleavage nucleophile[4](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014). Within the recombination synapse, only two of the four potential active sites are active at any one time, the "half-of-the-sites" activity that accounts for the two-step mechanism[4](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014).
**Simple reaction requirements.** FLP, like Cre, is not particular about DNA topology or target-site orientation. It acts on supercoiled or nicked circles as well as linear molecules and promotes both intra- and intermolecular reactions: inverted sites give DNA inversion, direct sites give deletion[4](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014). Binding of FLP to the FRT site also bends the DNA, and mutants defective in bending have been isolated[8](https://pubmed.ncbi.nlm.nih.gov/2648010/).
## Use in genome engineering
Because FLP rearranges DNA precisely between engineered FRT sites, it became one of the standard tools of conditional genetics, alongside Cre-loxP from bacteriophage P1 and later Dre-rox; all three systems use the same basic mechanism, differing in recombinase and recognition site[6](https://digitalcollections.ohsu.edu/record/42751/files/Jahncke.Jennifer.2024.pdf).
**Stable cell lines.** The commercially available Flp-In system places a single FRT site in the genome of a parental cell line; a gene-of-interest construct carrying an FRT site is then integrated at that locus by FLP-mediated recombination (O'Gorman et al., 1991)[9](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/flpinsystem_man.pdf). Because the FRT site occupies the same genomic location in every clone, derived cell lines are isogenic, which reduces the variability that random integration introduces[10](https://livrepository.liverpool.ac.uk/3185193/1/201443648_April2024.pdf). A limitation of such approaches is that the recombination event can integrate entire vector sequences, including the vector backbone, into the FRT site[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC9175168/). Recombinase-mediated cassette exchange (RMCE) refines this by exchanging a defined cassette between two FRT sites, giving predictable expression from a pre-characterized locus[12](https://www.sciencedirect.com/science/article/pii/S0022283611000210).
**Conditional genetics in animals.** FLP-FRT was described at roughly the same time as Cre-lox, but the native FLP enzyme is labile at mammalian body temperatures, which limited its efficiency in mammals[5](https://www.jax.org/news-and-insights/jax-blog/2014/November/going-beyond-cre-lox). Its optimum recombination activity in mammalian cells is at 30 °C, with relatively low activity at 37 °C, consistent with its physiological role in yeast[12](https://www.sciencedirect.com/science/article/pii/S0022283611000210). Buchholz and colleagues improved activity above the 30 °C optimum using error-prone PCR to introduce point mutations followed by [DNA shuffling](https://www.edgepedia.org/dna-shuffling) and in vivo selection, producing the enhanced variant FLPe[12](https://www.sciencedirect.com/science/article/pii/S0022283611000210) • [3](https://pmc.ncbi.nlm.nih.gov/articles/PMC1764711/). FLPe works more efficiently in mice and mammalian cells, though FLP-FRT still generally remains less efficient than Cre-lox; its most common use is to remove FRT-flanked selection cassettes from loxP-containing targeting constructs used to make Cre-lox mice[5](https://www.jax.org/news-and-insights/jax-blog/2014/November/going-beyond-cre-lox). Because FLP and Cre recognize different sites, the two systems can be combined in one animal for dual or sequential recombination events, and intersectional approaches can require co-expression of multiple recombinases to achieve greater cellular specificity[6](https://digitalcollections.ohsu.edu/record/42751/files/Jahncke.Jennifer.2024.pdf).
**Plants and marker removal.** [Site-specific recombination](https://www.edgepedia.org/site-specific-recombination) systems are used in plant transgenesis both to eliminate antibiotic marker genes and to place a target site in the genome so a gene-of-interest can be inserted precisely. In one scheme, heat activation of the FLP recombinase drives recombination between two collaterally oriented FRT sites, excising the antibiotic marker gene lying between them[13](https://www.fao.org/4/k3334e/k3334e.pdf).
## Comparison with Cre-lox and practical limits
The two systems are mechanistically parallel: a trans-acting recombinase and a short recognition site, with the identity and orientation of the sites determining whether excision, inversion, or insertion results[6](https://digitalcollections.ohsu.edu/record/42751/files/Jahncke.Jennifer.2024.pdf). Cre-lox is generally the more efficient choice in mammalian cells, while FLP-FRT's independence from Cre-lox is its main practical advantage, allowing the two to be run concurrently in the same cell or animal[5](https://www.jax.org/news-and-insights/jax-blog/2014/November/going-beyond-cre-lox) • [6](https://digitalcollections.ohsu.edu/record/42751/files/Jahncke.Jennifer.2024.pdf). In mammalian applications, bacterial sequences carried on the integrating vector can be CpG-rich and vulnerable to methylation, which can silence a reporter, and the integration of vector backbone noted above can complicate the outcome[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC9175168/).
## References
1. [McLeod M., Craft S., Broach J.R. (1986). Identification of the crossover site during FLP-mediated recombination in the *Saccharomyces cerevisiae* plasmid 2 microns circle. Molecular and Cellular Biology 6(10):3357–3367.](https://pmc.ncbi.nlm.nih.gov/articles/PMC367081/)
2. [Resolution of immobile chi structures by the FLP recombinase of 2 microns plasmid. Journal of Molecular Biology (1994). PMID 7932750.](https://pubmed.ncbi.nlm.nih.gov/7932750/)
3. [High-Efficiency FLP and ΦC31 Site-Specific Recombination in Mammalian Cells. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC1764711/)
4. [Jayaram M. et al. (2015). An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective. Microbiology Spectrum.](https://journals.asm.org/doi/10.1128/microbiolspec.mdna3-0021-2014)
5. [Going beyond Cre-lox. JAX Blog (2014).](https://www.jax.org/news-and-insights/jax-blog/2014/November/going-beyond-cre-lox)
6. [Jahncke J. (2024). Conditional genetics systems including Flp-FRT and Dre-rox. OHSU digital collections.](https://digitalcollections.ohsu.edu/record/42751/files/Jahncke.Jennifer.2024.pdf)
7. [The 2 micron plasmid of *Saccharomyces cerevisiae*: A miniaturized selfish genome with optimized functional competence. Cell Cycle (2013).](https://www.sciencedirect.com/science/article/pii/S0147619X13000292)
8. [FLP recombinase of the 2 microns circle plasmid of *Saccharomyces cerevisiae* bends its DNA target. (1989). PMID 2648010.](https://pubmed.ncbi.nlm.nih.gov/2648010/)
9. [Flp-In System manual, Thermo Fisher Scientific.](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/flpinsystem_man.pdf)
10. [University of Liverpool thesis (2024) on stable cell line generation using FLP-mediated recombination.](https://livrepository.liverpool.ac.uk/3185193/1/201443648_April2024.pdf)
11. [Expression of mammalian proteins for diagnostics and therapeutics: a review. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC9175168/)
12. [Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges. Journal of Molecular Biology (2011).](https://www.sciencedirect.com/science/article/pii/S0022283611000210)
13. [Elimination of marker genes and targeted integration of transgenes via the FLP/FRT-recombination system. FAO.](https://www.fao.org/4/k3334e/k3334e.pdf)
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*Topic: Encyclopedia › Life and health › Biological foundations › Genetics and genomic reference › Mobile genetic elements and plasmids*
*Initially written Sep 17, 2026 · Reviewed: Oct 21, 2026 · Edited: Oct 15, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*
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