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lox stop lox Cre-lox Plasmids


Background

The Cre-lox system, derived from P1 bacteriophage, is a potent and specific system for controlling gene expression. The protein Cre recombinase recognizes 34 bp loxP sites, and the orientation and location of the loxP sites determines how the genetic material will be rearranged. The schematic below shows the three types of rearrangements: inversion, deletion and translocation.

  • Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination results in an inversion and the region of DNA between the loxP sites is reversed.
  • Deletion: If the sites face in the same direction, the sequence between the loxP sites is excised as a circular piece of DNA (and is not maintained).
  • Translocation: If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites.
recombination outcomes from loxp site orientation

For a more thorough introduction, check out Addgene’s Cre-lox blog post.

Based on these Cre-lox recombination principles, scientists have developed constructs to activate/inactivate genes when Cre is present. By expressing Cre at specific times or locations, you can precisely control expression of your gene of interest. Many Cre constructs also contain fluorescent labels that indicate if recombination has occurred, allowing for direct comparison of Cre+ and Cre- cells. While Cre-lox recombination is a very useful tool, Cre expression can be toxic in some systems, notably Drosophila. Read Addgene's Blog on Cre alternatives from Gerald Rubin's lab.

Cre-containing Plasmids

Scientists have developed ways to tightly control Cre expression and to optimize Cre expression once it is induced. Addgene has a wide variety of Cre recombinase plasmids. Special features include:

  • Inducible Cre: These constructs require the addition of an exogenous ligand (e.g. tamoxifen) to activate Cre. One advantage of this system is tight temporal regulation.
  • Promoter-regulated Cre: The promoter region defines the areas in which Cre will be expressed. Cre may be expressed globally under a broadly active promoter like CAG, or expressed only in a subset of cells under a more specific promoter (e.g. Rho-Cre is expressed in the retina).
  • Fluorescent Cre: The fusion of Cre to a fluorescent reporter enables visualization of Cre expression.
  • Optimized Cre: Codon-optimized Cre (iCre) is expressed at higher levels in mice than P1 bacteriophage Cre, facilitating high rates of Cre-driven recombination. CREM (or Cre-M) has been engineered to contain an intron, preventing Cre expression when cloning in E. coli. This alteration enables the generation of a single vector containing Cre and a floxed construct (unmodified Cre will cause recombination in E. coli, deleting the floxed portion of a construct during the cloning process).

Use the search box below to find the perfect Cre expression construct for your experiment. You can search the table for the promoter, fusion, or expression system of choice.

   ID Plasmid Description Promoter Expression System PI
13776 pCAG-Cre:GFP Cre-GFP fusion CAG Mammalian Cepko
14797 pCAG-CreERT2 Cre-ERT2 - Tamoxifen inducible CAG Mammalian Cepko
13777 pCAG-ERT2CreERT2 ERT2-Cre-ERT2-Tamoxifen inducible CAG Mammalian Cepko
13775 pCAG-Cre Cre-Myc CAG Mammalian Cepko
26646 pCAG-Cre-IRES2-GFP Cre and GFP coexpression CAG Mammalian Chenn
12168 pMB80 (R26-CreER) CreERT2 with loxp cassette; Targeting vector for Rosa26 locus Mammalian Jacks
11918 pBS513 EF1alpha-cre Cre EF-1 alpha Mammalian Sauer
11916 pBS185 CMV-Cre Cre hCMV Mammalian Sauer
11923 pBS598 EF1alpha-EGFPcre Cre-EGFP fusion EF1 alpha Mammalian Sauer
12238 pLOX-CW-CRE Cre CMV Lentiviral Trono
12265 pHR-CMV-nlsCRE Cre CMV Lentiviral Trono
12106 LV-Cre Cre CMV Lentiviral Verma
27546 pLM-CMV-R-Cre Cre and mCherry coexpression CMV Lentiviral Sadelain
17408 Puro.Cre empty vector Cre, Puro resistance and miRNA expression PGK Lentiviral Jacks
22776 MSCV CreERT2 puro Cre-ERT2 - tamoxifen inducible Retroviral Jacks
27321 pENTR/D_creERt2 creERt2 - tamoxifin inducible; Gateway entry vector none Entry Vector Zon
13763 pTriEx-HTNC Cre fused to HIV-TAT - promotes cellular uptake of recombinant Cre Bacterial Rajewsky
47551 pDD104 (Peft-3::Cre) Cre eft-3 C. elegans Goldstein
26745 pDIRE iCre and FlpO, required for dual RMCE EF1alpha Mammalian Zeller
15028 Hsp68-CreER(TM) (DM#264) CRE-ER(TM) - tamoxifen inducible Hsp68 Mammalian Melton
11957 pBS595 tet-hCMV-EGFPcre Cre-EGFP fusion Tet inducible Mammalian Sauer
24704 GFAP-Cre Cre GFAP Mammalian Sofroniew
26889 pmCherry-CIBN-CreC CIBN-CreC and mCherry coexpression; Light inducible; Use with plasmid 26888. Mammalian Tucker
26888 pmCherry-CRY2-CreN CRY2-CreN and mCherry coexpression; Light inducible; Use with plasmid 26889. Mammalian Tucker
25997 LV-Cre pLKO.1 Cre and shRNA coexpression CMV Lentiviral Fuchs
30205 Cre-IRES-PuroR Cre and PuroR coexpression EF1alpha Lentiviral Kotton
24593 AAV-pgk-Cre codon optimized Cre PGK AAV Aebischer
13779 pRho-Cre Cre-Myc rhodopsin Mammalian Cepko
14796 pClusterin-ERT2CreERT2 ERT2-Cre-ERT2 - Tamoxifen inducible. Expressed in Muller glia in the retina. Clusterin Mammalian Cepko
8395 p210 pCMV-CREM CREM CMV Mammalian Green
60820 pSECC Cre, Cas9 and sgRNA expression EF1alpha Lentiviral Jacks
24064 MSCV-Cre Cre and EGFP coexpression Retroviral Littman
34565 pMSCVhygro-Cre Cre and HygroR coexpression Retroviral Ge
48201 CAG-GFP-IRES-CRE Cre and GFP coexpression CAG Retroviral Gage
49054 CAG-GFP/cre Cre-GFP fusion CAG Retroviral Gage
49056 AAV-GFP/Cre Cre-GFP fusion CMV AAV Gage
60229 AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR Cre and sgRNA coexpression Cbh AAV Zhang
26850 pBF3038 Cre codon optimized for yeast Gal1 Yeast Sandmeyer
35619 pTAT-CRE Cre fused to HIV-TAT Bacterial Dowdy
31132 pCSCre Cre CMV Xenopus Ryffel
13780 pNrl-Cre Cre-Myc, Expressed in rod photorecetor cells in the retina Nrl Mammalian Cepko
50797 pUAS-Cre Cre UAS/Hsp70 Mammalian Cepko
32606 Ttr:Cre Cre expressed in visceral endoderm MCK Mammalian Hadjantonakis
59720 Nanog-CreER targeting construct creERT2; Targeting vector for Nanog locus mNanog Mammalian Hanna
12529 pBS MCK CRE Muscle-specific expression of Cre MCK Mammalian Kahn
40591 hGFAP-Cre mouse astrocyte expression of Cre hGFAP Mammalian Messing
45359 pNK-TGCK Cre-EGFP fusion; Tet inducible - rrTA expression driven by mouse Nkx cardiac enhancer and promoter fragment minimal TetO-CMV Mammalian Wu
62219 Nxph4-2A-CreERT2 Targeting Vector CreERT2 - Tamoxifin inducible; Targeting vector Mouse Nxph4 Mammalian Zeng
50935 MSCV-PIG-Cre Cre and GFP coexpression PGK Retroviral Pandolfi
59701 pRetroQ-Cre-ERT2 Cre-ERT2 - tamoxifen inducible cmvIE Retroviral Youle
51507 AAV pmSyn1-EBFP-Cre Cre-EBFP fusion; Expression in neurons. synapsin AAV Zeng
51904 paavCAG-iCre iCre CAG AAV Kim
55635 pAAV-EF1a-sCre sCre EF1alpha AAV Deisseroth
55636 pAAV-EF1a-Cre Cre EF1alpha AAV Deisseroth
55632 pAAV-Ef1a-mCherry-IRES-Cre Cre and mCherry coexpression EF1alpha AAV Deisseroth
55638 pAAV-EF1a-vCre vCre EF1alpha AAV Deisseroth
49455 pSH62-EBD Cre-EBD - Estradiol-dependent control Gal1 Yeast Gartenberg
60931 pPL5608_TEF1*-Cre_TRP1 Cre expressed at low levels mutant TEF1 Yeast Piper
13962 pBS mPrx1 Cre (CT#644) Cre; expression in developing limb mouse Prx1 Bacterial Tabin
36915 pJJH1320 Cre Bacterial Heinisch
45863 pCM157 Cre lac Bacterial Lidstrom
47117 pT3TS-Cre for in vitro transcription of Cre lac Bacterial Ekker
31309 pCARCre Cre cardiac actin Xenopus Ryffel
30524 pCSHSP:Cre heat shock inducible Cre Xenopus hsp70 Xenopus Ryffel
30525 pBSHSP:Cre;CMV:tdTomato-SceI Cre and tdTomato coexpression Xenopus hsp70 Xenopus Ryffel
24333 ins-cre_cryaa-Venus Cre-Venus fusion insulin Zebrafish Stainier
32144 pJFRC170-3XUAS-IVS-Cre::PEST Cre expressed at low levels to reduce toxicity; See PI page for other recombinases that are less toxic in Drosophila hsp70 Insect Rubin
33344 pLEW100cre-EP1-6G Cre T. brucei Cross
61391 pCS2-Cre.zf1 Cre codon modified for zebrafish CMV Zebrafish Burgess
37404 Cre Shine Cre::2A::targeted FP construct CMV Mammalian Hughes
11543 pPGK-Cre-bpA Cre PGK Mammalian Rajewsky
8405 p240 pPr-CREM/Ins CREM with insulators probasin Mammalian Green
11955 pBS505 EF1alpha-EGFPcre* Cre-EGFP fusion EF-1 alpha Mammalian Sauer
11919 pBS448 RSV-GFPcre Cre-GFP fusion Rous sarcoma virus (RSV) LTR/promoter Mammalian Sauer
11956 pBS594 promoterless EGFPcre Cre-EGFP fusion in a promoterless vector with several available restriction sites none Mammalian Sauer
11920 pBS500 EF1alpha-GFPcre Cre-GFP fusion EF1alpha Mammalian Sauer
26647 pCAG-Cre Cre CAG Mammalian Chenn
34564 pMSCVpuro-Cre Cre 5'LTR Retroviral Ge
60231 AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR Cre, KASH-tagged EGFP, and sgRNA expression hSyn AAV Zhang
60226 AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR Cre, luciferase, and sgRNA expression EFS AAV Zhang
26853 pBF3060 Cre codon optimized for yeast Gal1 Yeast Sandmeyer
19131 pIC-Cre Cre Bacterial Rajewsky
8404 p236 pPr-CREM CREM probasin Mammalian Green
8408 p255 pPr-CREM/Ins/CMV-STOP-luc CREM and CMV-STOP-luc cassette, separated by insulator probasin Mammalian Green
8394 p209 pCMV-cre-K Cre-K CMV Mammalian Green
8401 p224 pCMV-RFP/CREM floxed RFP within CREM CMV Mammalian Green
8403 p153 pPr-cre Cre probasin Mammalian Green
8406 p252 pPr-CREM/CMV-STOP-luc CREM and CMV-STOP-luc cassette probasin Mammalian Green
12494 p260 pCMV-CRE-M-AR(wt) CREM fused to AR ligand binding domain CMV Mammalian Green
12496 p270 pCMV-CRE-M-AR(LNCaP) CREM fused to mutant AR ligand binding domain CMV Mammalian Green
12493 p259 pCMV-CRE-M (BglII) CREM CMV Mammalian Green
12497 p274 pCMV-CRE-M-AR(T) CREM fused to mutant AR ligand binding domain CMV Mammalian Green
11958 pBS532 promoterless GFPcre promoterless CRE-GFP fusion none Mammalian Sauer
11961 pBS596 tet-hCMV-GFPcre tet inducible Cre-GFP fusion, metallothionein MT-I region including the polyadenylation site and several introns tet-hCMV Mammalian Sauer
11960 pBS537 tet-hCMV-GFPcre tet inducible Cre-GFP fusion tet-hCMV Mammalian Sauer
27493 pBT140 Cre CMV Mammalian Luo
34568 WZLneo-Cre Cre 5'LTR Retroviral Ge
60932 pPL5606_TEF1*-Cre_ADE2 Cre expressed at low levels mutant TEF1 Yeast Piper
60930 pPL5071_TEF1*-Cre_URA3 Cre expressed at low levels mutant TEF2 Yeast Piper
60933 pPL5628_TEF1*-Cre_MET15 Cre expressed at low levels mutant TEF3 Yeast Piper
15509 pBS-M71-IRES-CreFNF M71 targeting vector with IRES CreFNF (neo-selectable marker flanked by FRT sites) Mammalian Mombaerts
15508 pBS-OMP-CreFNF OMP targeting vector with Cre FNF (neo-selectable marker flanked by FRT sites) Mammalian Mombaerts
17736 pOG231 NLS-Cre CMV Mammalian Wahl
33342 LGmCreER (non-self deleting) CreERT2 PGK Retroviral Lowe
66691 FUG-T2A-cre GFP-T2A-Cre Lentiviral Luikart
69570 pAAV-EF1a-N-CretrcintG Cre recombinase dependent on GFP (CRE-DOG) EF1alpha AAV Cepko
69571 pAAV-EF1a-C-CreintG Cre recombinase dependent on GFP (CRE-DOG) EF1alpha AAV Cepko
69572 pCAG-N-CretrcintG Cre recombinase dependent on GFP (CRE-DOG) CAG Mammalian Cepko
69573 pCAG-C-CreintG Cre recombinase dependent on GFP (CRE-DOG) CAG Mammalian Cepko
72256 pCDH-CB-copGFP-T2A-iCre copGFP and iCre CB Lentiviral Oka
46389 pArcCreER CreERT2;Targeting vector for Arc locus Mammalian Luo
46388 pFosCreER CreERT2;Targeting vector for Fos locus Mammalian Luo
68544 AAV-Cre-GFP Cre CMV AAV Nestler
33340 LGmCreER (self deleting) CreERT2 - tamoxifin inducible PGK Retroviral Lowe
51263 hGFAP-Roxed-Cre Dre-dependent Roxed-Cre GFAP Mammalian Pelczar
66690 Fuc-T2A-cre mCherry-T2A-Cre Lentiviral Luikart
51276 Thy1.2-Roxed-Cre Dre-respondent Cre Thy1 Mammalian Pelczar
51273 pCAG-Roxed-Cre Dre-respondent Cre CAG Mammalian Pelczar
51267 pCAG-Co-InCreN C-terminal component of the Co-InCre system CAG Mammalian Pelczar
51268 pCAG-Co-InCreC N-terminal component of the Co-InCre system CAG Mammalian Pelczar
33345 pLEW100cre-EP1 Cre Trypanosoma brucei Cross
64770 pSS146 Cre-EBD78 Tdh4 Yeast van Leeuwen
68468 Cas9-2A-Cre Cas9 and Cre CAG Mammalian Zhang
62730 CRE recombinase Cre T7 Bacterial Geijsen
61570 Pvalb-2A-Cre targeting vector Cre; Targeting vector for Pvalb locus Mammalian Zeng
61574 Slc17a7-IRES2-Cre targeting vector Cre; Targeting vector for Slc17a7 locus Mammalian Zeng
62939 pET-Cre-6xHis Cre-6xHis T7 Bacterial Liu
61573 Rasgrf2-2A-dCre targeting vector DHFR-destabilized Cre; targeting vector for Rasgrf2 Mammalian Zeng
63704 pRetroX GFP T2A Cre GFP T2A Cre tight TRE Retroviral Foijer
65795 pBBI iCre CMV Mammalian Ohtsuka
62657 pAYC iCre CAG Mammalian Ohtsuka
64769 pTW040 Cre-EBD78 TDH3 Yeast van Leeuwen
66692 pRubiC-T2A-cre mCherry-T2A-Cre expression in newborn neurons Retroviral Luikart
66693 pRubiG-T2A-cre GFP-T2A-cre expression in newborn neurons Retroviral Luikart
69916 pAAV.cTNT.iCre iCre and tdtomato Chicken cardiac troponin T AAV Pu
49111 pEMS1980 iCre with MCS for inserting promoter none AAV Simpson
60877 pMAZe Cre and dtomato hsp70 Zebrafish Lewis
65727 pcDNA3.1-CMV-CFP;UBC-Cre25nt Cre with a 25 nucleotide extracellular vesicle targeting sequence and CFP UBS Mammalian van Rheenen
68448 iRFP670, Cre, and GFP iRFP670, Cre, and GFP EFS Lentiviral Jacks
70120 pEMS2159 iCre with MCS for inserting promoter, WPRE none AAV Simpson
72257 pCDH-CB-iCre iCre CB Lentiviral Oka
73350 pAdx-CMV-iCre-P2A-copGFP iCre and GFP CMV Adenoviral Oka
73472 RabV CVS-N2c(deltaG)-mCherry-P2A-Cre Cre and mCherry for monosynaptic circuit mapping and genetic manipulation Neurotropic virus Jessell
73474 RabV CVS-N2c(deltaG)-Cre Cre for monosynaptic circuit mapping and genetic manipulation Neurotropic virus Jessell
73717 pMLS328 Cre eft-3 C. elegans Jorgensen

loxP (floxed) Constructs

These constructs allow for Cre-regulated gene expression. Depending on the construct, Cre may activate or repress gene expression. Common construct types include:

  • Cre-dependent Gene Expression: Placing a stop codon with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette) 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.
  • Cre-dependent Gene Knockout: Conversely, putting 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.
  • Cre-dependent 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.

Empty backbones to regulate your gene of interest:

ID Plasmid Description Expression System PI
11578 pSico Cre activates shRNA expression, removal of EGFP; See also similar plasmids pSico PGK GFP and pSico PGK puro Lentiviral Jacks
11579 pSicoR Cre turns off shRNA expression, removal of EGFP; See also similar plasmids pSicoR PGK puro, pSicoR-Ef1a-mCh-Puro, pSicoR-Ef1a-mCh and pSicoR-mCh-empty Lentiviral Jacks
11584 Lox-Stop-Lox TOPO Cre activates gene of interest by removing Stop sequences; Puro selection. See plasmid 35688 for Hygro selection. Mammalian Jacks
11925 pBS302 Cre activates gene of interest by removing Stop sequences. Can be used for injection into fertilized zygotes Mammalian Sauer
21270 pBigT Cre activates gene of interest by removing neoR and stop sequence; used for making transgenic mice. Plasmid 15037 also contains IRES-EGFP Mammalian Costantini
32702 pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE Cre activates your gene fused to eGFP, removes dsRed. See article for other similar plasmids. Retroviral Clevers
15912 CTV Cre activates gene of interest, removes Neo and stop cassette; Contains flanking arms for Rosa26 integration; See similar plasmid 11739 Mouse Targeting Rajewsky
11072 p079 pPNT6 Cre activates gene of interest, removes Neo cassette. Used in gene targeting. Mouse Targeting Shivdasani

Cre Reporters and Tools

In this subtype of loxP plasmids, reporter genes indicate which cells have undergone Cre recombination. These plasmids can be useful for lineage tracing, evaluating infection/transfection efficiency, selecting desired clones, and making transgenic mice.

ID Plasmid/Article Description Expression System PI
See article Brainbow constructs Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors Mammalian Sanes
See article Multibow constructs Multibow uses a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination to maximize label diversity Mammalian Megason
13769 pCALNL-DsRed Cre dependent DsRed expression Mammalian Cepko
13770 pCALNL-GFP Cre dependent GFP expression Mammalian Cepko
8389 p212 pCMV-EGFP/RFP EGFP-dsRed gene switch plasmid Mammalian Green
22799 Ai9 Rosa26 targeting vector, Cre dependent tdtomato expression. See article for more colors. Mammalian Zeng
24334 hsp70l-loxP-mCherry-STOP-loxP-H2B-GFP_cryaa-cerulean Heat-inducible reporter with Cre dependent H2B-RFP expression Zebrafish Stainier
51269 pCAG-loxPSTOPloxP-ZsGreen Cre dependent ZsGreen expression Mammalian Pelczar
37402 Cre Stoplight 2.4 Zsgreen is expressed in cells without Cre, mCherry is expressed with Cre Mammalian Hughes
16422 betaRBPIG Double fluorescent, double selectable cre/loxP reporter Mammalian Hescheler
12463 p231 pCMVe-betaAc-STOP-luc Cre dependent luciferase expression Mammalian Green
32145 pJFRC172-10XUAS-loxP>-dSTOP-loxP>-myr::GFP Cre dependent myr-GFP expression Insect Rubin
60877 pMAZe Reporter construct for lineage tracing and mosaic analysis in zebrafish Zebrafish Lewis

References

  • Bacteriophage P1 Site-Specific Recombination. I. Recombination Between loxP Sites. Sternberg N, Hamilton D. J Mol Biol. 1981 Aug 25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed.

  • Site-Specific DNA Recombination in Mammalian Cells by the Cre Recombinase of Bacteriophage P1. Sauer B, Henderson N. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166-70. PubMed.

  • Targeted Oncogene Activation by Site-Specific Recombination in Transgenic Mice. Lakso M, Sauer B, Mulder KL, Westphal H. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6232-6. PubMed.

  • Cre-lox-Regulated Conditional RNA Interference from Transgenes. Ventura A, Meissner A, Jaenisch R, Jacks T. Proc Natl Acad Sci U S A. 2004 Jul 13;101(28):10380-5. doi:10.1073/pnas.0403954101. Epub 2004 Jul 6. PubMed.

  • Controlled Expression of Transgenes Introduced by In Vivo Electroporation. Matsuda T, Cepko CL. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1027-32. doi: 10.1073/pnas.0610155104. Epub 2007 Jan 5. PubMed.

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