Cre-Lox and Other Site-Specific Recombinases

Site-specific recombinase (SSR) systems derived from fungi and bacteriophages are powerful genome engineering tools used to precisely manipulate DNA and control the expression of specific genes in cells or organisms. Each recombinase enzyme recognizes and binds pairs of short, specific DNA target sites, and catalyzes recombination between them. Most SSR enzymes are categorized into two distinct families, serine recombinases and tyrosine recombinases, based on sequence similarity and the amino acid residue used in the active site.

PhiC31 integrase, derived from a Streptomyces phage, and Bxb1 integrase, from a mycobacteriophage, are serine SSRs that cleave all four DNA strands at once. They can mediate the integration of a large DNA payload containing an attB target site into a genomic attP site. So far, their practical application has been limited by low integration efficiency but research groups continue to optimize these tools.

The best characterized SSR systems, Cre and Flp, are tyrosine recombinases derived from P1 E. coli bacteriophage and S. cerevisiae, respectively. Other tyrosine recombinases include Dre, R, Nigri, SCre, VCre, and Vika. These systems work by sequential strand exchange at a specific target site (called loxP for Cre, or FRT for Flp). The nature of the DNA rearrangement after recombination (inversion, deletion, or translocation) depends on the orientation of each target site (see Figure 1).

Figure 1: Recombination outcomes are determined by target site orientation. A) If the target sites (loxP) are on the same DNA strand in opposite directions, recombination results in an inversion and the region of DNA between the target sites is reversed. B) If the target sites are on the same DNA strand (in cis) in the same direction, the sequence between the sites is excised as a circular piece of DNA (and is not maintained). C) If the sites are on separate DNA molecules (in trans), a translocation event is generated between the target sites. Created with BioRender.com.

Scientists have adapted these systems to activate/inactivate genes when a recombinase enzyme is present. Read on to learn about the different ways that Cre-lox and other site-specific recombinases can be used to modify the genome or control gene expression or skip ahead to browse highlighted plasmids expressing Cre, Flp, Dre, other recombinases, as well as useful empty backbones or reporters.

Experimental Considerations

Recombinase Recognition Sites

Each enzyme functions analogously but interacts only with their own target sites (for example, Cre with lox sites, Flp with FRT sites, Dre with rox sites), enabling precise genetic manipulation without cross-reactivity with other recombinase systems.

Target Site	Canonical Sequence	Structure (Length)	Common Variants
Cre	loxP	ATAACTTCGTATAgcatacatTATACGAAGTTAT	13 bp inverted repeats + 8 bp spacer (34 bp)	lox2272, lox5171, lox511, loxN, lox66/lox71
Dre	rox	TAACTTTAAATAATgccaATTATTTAAAGTTA	14 bp inverted repeats + 4 bp spacer (32 bp)	Currently, only the canonical rox site is widely used.
Flp	FRT	GAAGTTCCTATTCCGAAGTTCCTATTCtctagaaaGTATAGGAACTTC	13 bp inverted repeats + 8 bp spacer (48 bp or 34 bp)	F3, F5, FRT14, FRT15
	minimal FRT	GAAGTTCCTATTCtctagaaaGTATAGGAACTTC

Table 1. Summary of Cre, Dre, and Flp Recognition Sites.

Controlling Recombinase Expression

By expressing the enzyme at specific times or locations using cell-specific promoters or inducible systems, you can precisely control and optimize expression of your gene of interest.

• Inducible: The recombinase may be fused to a ligand-binding domain such as the human estrogen receptor (ERT2) so that the enzyme is only activated in the presence of tamoxifen. A tetracycline-regulated or other drug-inducible approach may also be used.
• Split proteins: The recombinase is split into inactive N- and C-terminal fragments and placed under the control of different promoters. Expression of both fragments in the same cell reconstitutes a functional protein, allowing recombination of the DNA between target sites.
• Codon optimization: Modifying the DNA sequence of the recombinase to align with your organism's preferred codon usage will improve expression. For example, iCre or FLPo in mammalian cells may facilitate higher rates of Cre-driven recombination.

Gene Expression Strategies

Depending on how the target sites are arranged, recombinases can be used for more than inversion, deletion, or translocation of a gene of interest. Using Cre or Flp as examples, here are a few popular recombinase-dependent gene expression strategies.

• Gene Expression: A stop codon flanked with loxP sites (often called a "lox-STOP-lox" or "LSL" cassette) placed upstream of a gene of interest will prevent gene expression in the absence of Cre. In the presence of Cre, the stop codon is excised, and gene expression proceeds.
• Gene Knockout: Placing the loxP sites on either side of a gene (called "floxing", for "flanked by loxP"), will permit gene expression until Cre is present, at which time the gene will be disrupted or deleted.
• shRNA Expression: Cre-lox can be used to turn shRNA constructs on or off. In floxed-shRNA constructs, Cre can excise the shRNA to return gene expression to physiological levels. In lox-STOP-lox shRNA constructs, Cre expression promotes shRNA expression.
• Gene Switch: These constructs contain two genes of interest, Genes A and B. When Cre is absent, only Gene A is translated correctly. Cre expression excises Gene A and alters the reading frame to allow in-frame translation of Gene B.
• FLEx Switch (for “flip-excision”): Also known as DIO (Double-floxed Inverse Open reading frame), this system allows scientists to utilize recombination elements such as Cre to turn off the expression of one gene while simultaneously turning on another. The FLEx switch system takes advantage of the orientation specificity of Cre and the different types of target sites available, both mutant and wild type. The ability to manipulate the number, orientation, and type of target sites that flox your genes of interest makes FLEx switch a powerful experimental tool. For more information on FLEx switch and what you can do with it, read Addgene's blog on FLEx Vectors.
• Flp-IN: A single FRT site within the genome of a host cell line serves as the target location for integration of a plasmid containing a gene of interest and another FRT site. Flp recombinase recognizes and mediates recombination between the two FRT sites, resulting in the stable integration of the gene of interest into the genome at the defined FRT site.
• Multiplexing: Orthogonal recombinase systems can be combined in multiple, independent recombination events in a single experimental system, enabling multi-layered genetic control.
• Recombinase-mediated cassette exchange (RMCE): When matching but mutually incompatible recognition sites flank both a DNA target site and an exogenous donor cassette, the donor cassette replaces the target site cassette in a double-crossover event.

Toxicity

Cre-lox recombination is a very useful tool, but Cre expression can be toxic in some systems, such as Drosophila and mouse, particularly in proliferating cells. Expression may cause repetitive recombination leading to genomic instability and cell death. It is important to incorporate appropriate controls such as Cre-only lines to account for cellular toxicity in your system. Using inducible Cre systems in combination with lineage-specific drivers can also help reduce off-target events and the systemic effects of Cre toxicity.

Leakiness

Leaky expression, where the gene of interest is expressed even in the absence of the recombinase, and spontaneous recombination between target sites are common concerns when using Cre or Flp-dependent expression systems and may affect the interpretation of your experimental results. Attenuated iCre(R297T), a point mutant iCre variant, reduces the number of recombination events but can improve specificity in targeted neuronal populations. Read our blog post about how to minimize leakiness in experiments using AAV vectors.

Highlighted Plasmids

Browse the tables below to find popular plasmids expressing your favorite site-specific recombinase (Cre, Flp, Dre, other recombinases). Use the search bars or sort buttons to find plasmids based on your expression system of choice, promoter, or search by inducible system (e.g., search for "split", "rapamycin", “tamoxifen”, or “light”):

Cre Recombinase Plasmids

Additional Addgene resources

• Explore our full catalog for plasmids expressing Cre recombinase and filter the results by expression system and vector type.
• Find in-stock viral preps expressing Cre and other site-specific recombinases.
• Many of our in-stock viral preps feature Cre-dependent tools, or consider our AAV Packaged on Request service for more options.

Addgene blog

• Plasmids 101: Cre-lox
• Cre-ating New Methods for Site-specific Recombination in Drosophila
• Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP
• Plasmids 101: FLEx Vectors
• Optimizing AAV DIO and FLEx Vector Expression
• Plasmids 101: Knockout/Knock-In Plasmids
• Advanced Uses of Cre-lox and Flp-FRT — A Neuroscientist’s View

Flp Recombinase Plasmids

Additional Addgene resources

• Search our full catalog for plasmids expressing Flp recombinase and filter the results by expression system and vector type.
• Find in-stock viral preps expressing Flp and other site-specific recombinases.
• Many of our in-stock viral preps feature Flp-dependent tools, or consider our AAV Packaged on Request service for more options.

Addgene blog

• Advanced Uses of Cre-lox and Flp-FRT — A Neuroscientist’s View

Dre Recombinase Plasmids

Additional Addgene resources

• Search our full catalog for plasmids expressing Dre recombinase and filter the results by expression system and vector type.
• Find in-stock viral preps expressing Dre and other site-specific recombinases.

Other Site-Specific Recombinase Plasmids

Recombinase-Dependent Empty Backbones

Find an empty backbone to regulate your gene of interest. Depending on the construct, gene expression may be activated or repressed. Search by recombinase (Cre, Flp, Dre, etc.) or target site (lox, FRT, rox, etc.)

Additional Addgene resources

• Use the target site sequence for your recombinase (see Table 1) to Search by Sequence to find other Cre-, Flp-, or Dre-dependent plasmids.

Recombinase-Dependent Reporter Plasmids

Incorporating a fluorescent protein into your expression vector allows you to visualize which cells have undergone recombination. Reporter plasmids can also be useful for lineage tracing, evaluating infection/transfection efficiency, selecting desired clones, and making transgenic mice.

Pooled Libraries

Pooled Library Name	Type	PI	Description
Mouse Tumor Suppressor Gene CRISPR Knockout Library	CRISPR	Sidi Chen	Use this AAV CRISPR knockout library expressing luciferase and Cre to interrogate tumorigenesis in vivo using a mouse line expressing Cre-dependent Cas9.

Kits

Kit Name	Type	PI	Description
dRMCE Plasmid Kit	Genome Engineering	Rolf Zeller	Use this dual recombinase-mediated cassette exchange (dRMCE) plasmid kit to re-engineer conditional alleles in mouse embryonic stem cells.

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Content last reviewed on 31 July 2025.