cre recombinase sequencecre recombinase sequence

Please sign back in to continue your session. Liang S, Wang Y, Kang M, Deng J, Chen L, Hong X, Hou FF, Zhang F. Front Cell Dev Biol. Cre-Lox recombination is commonly used to circumvent embryonic lethality caused by systemic inactivation of many genes. sharing sensitive information, make sure youre on a federal Tyr 324 acts as a nucleophile to form a covalent 3-phosphotyrosine linkage to the DNA substrate. The Cre protein (encoded by the locus originally named as "Causes recombination", with "Cyclization recombinase" being found in some references)[21][22] consists of 4 subunits and two domains: The larger carboxyl (C-terminal) domain, and smaller amino (N-terminal) domain. Regulation of its activity has been accomplished to impart spatial and/or temporal control over recombination of the target gene. This conserves the energy of the phosphodiester bond, allowing the reaction to be reversed without the involvement of a high-energy cofactor. Displays both strands of base paired nucleotide sequences with annotated enzymes, plasmid features, Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Learn more, Download our file to copy and paste plasmid data, Learn more about Addgene materials from user-contributed reports describing AAV and antibody experiments, Basic analysis for a user-entered sequence; includes restriction sites and map, Digital collection of empty plasmid backbones from publications and commercially available sources. File contains the nucleotide sequence and annotated features in GenBank flat file format. What strain of bacteria does my stab contain? Can Cre Recombinase be used to move an insert between expression vectors? Learn about the latest plasmid technologies and research tools. How do I prepare and deposit my plasmids? It was unclear whether only one allele in each T-cell or 50% of T cells had 100% deletion in both alleles. This website uses cookies to ensure you get the best experience. In particular, the clear demonstration of its usefulness in precisely defining the complex relationship between specific cells/circuits and behaviors for brain research,[12] has promoted the NIH to initiate the NIH Blueprint for Neuroscience Research Cre-driver mouse projects in early 2000. [3], Cre recombinase plays important roles in the life cycle of the P1 bacteriophage. EcoRI fragments of the P1 bacteriophage genome were generated and cloned into lambda vectors. Please enable it to take advantage of the complete set of features! [29] Failure to activate both recombination events simultaneously confounds the interpretation of cell fate mapping results. Once tamoxifen is introduced, it is metabolized into 4-hydroxytamoxifen, which then binds to the ER and results in the translocation of the CreER into the nucleus, where it is then able to cleave the lox sites. First, the nucleotide sequence identity in the spacer region of lox site. Empty backbones to regulate your gene of interest: In this subtype of loxP plasmids, reporter genes indicate which cells have undergone Cre recombination. Cells alter their phenotype in response to numerous environmental stimuli and can lose the expression of genes typically used to mark their identity, making it difficult to research the contribution of certain cell types to disease. The activity of the Cre enzyme can be controlled so that it is expressed in a particular cell type or triggered by an external stimulus like a chemical signal or a heat shock. In this fashion, opposite DNA strands are joined together. This reaction links the recombinase protein to the DNA via a phospho-tyrosine linkage. Like other members of the integrase family, Cre catalyzes recombination between two identical double-stranded DNA sites of a particular sequence ( 1 - 3 ). Annu Rev Biophys Biomol Struct. The C domain is similar in structure to the domain in the Integrase family of enzymes isolated from lambda phage. Fields, Pathways [2], The enzyme plays important roles in the life cycle of the P1 bacteriophage, such as cyclization of the linear genome and resolution of dimeric chromosomes that form after DNA replication. As alluded to above, the Cre recombinase catalyzes the site specific recombination event between two loxP sites, which can be located either on the same or on separate pieces of DNA. At a DNA level many types of manipulations can be carried out. In the lysogenic cycle the phage genome replicates with the rest of the bacterial genome and is transmitted to daughter cells at each subsequent cell division. Cre is a site-specific recombinase from bacteriophage P1. Use Basic Local Alignment Search Tool (BLAST) via the NCBI website to determine similarity between How does it work? Reagents: FastStart PCR Master (Roche) DNA (50ng/l) 5' primer (100 M) 3' primer (100 M) Sterile H2O Volume: 7.5l 1.5l 0.06l Dre has been recently shown to function in mice and may be suited to. Increasing the length of DNA leads to decreased efficiency of Cre/lox recombination possibly through regulating the dynamics of the reaction. There is a problem with the plasmid I received. Learn more, Download our file to copy and paste plasmid data, Learn more about Addgene materials from user-contributed reports describing AAV and antibody experiments, Basic analysis for a user-entered sequence; includes restriction sites and map, Digital collection of empty plasmid backbones from publications and commercially available sources. Table lists feature name, location, Systems, Research Clipboard, Search History, and several other advanced features are temporarily unavailable. Purification and properties of the Cre recombinase protein", "Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation", "The Cre recombinase cleaves the lox site in trans", "Genetically engineered mouse models in cancer research", "Spontaneous recombinase activity of Cre-ERT2 in vivo", "Mutants of Cre recombinase with improved accuracy", https://en.wikipedia.org/w/index.php?title=Cre_recombinase&oldid=1135615154, Short description is different from Wikidata, Articles with German-language sources (de), Creative Commons Attribution-ShareAlike License 4.0, This page was last edited on 25 January 2023, at 19:59. The crystal structure at 2.4 A resolution of Cre bound to a loxP substrate reveals an intermediate in the recombination reaction, in which a Cre molecule has cleaved the substrate to form a covalent 3'-phosphotyrosine linkage with the DNA. This tool is suitable for deleting antibiotic resistance genes, but above all it allows conditional knockouts that can be induced at specific times in the cell type of choice. How can I track requests for my plasmids? Cre . RuvC is just one of these Resolvases that have been isolated in bacteria and yeast. Looking for antibodies related to this plasmid? 250 mM NaCl PCR protocol This section describes the composition of the mix and cycling conditions used for genotyping. Cre catalyses a reversible, directional recombination between two 34 bp loxP sites that consist of a pair of 13 bp inverted repeats flanking an 8 bp asymmetrical spacer 26, 27, 28. DNA sequences found between two loxP sites are said to be "floxed". This website uses cookies to ensure you get the best experience. There is a problem with the plasmid I received. To save your cart and view previous orders, sign in to your NEB account. Save time and money by placing an order with NEB. This 3 OH group is highly unstable, and the strand on which it is present must find its complement. [15][16] As of February 2019, CreLox recombination is a powerful tool and is used in transgenic animal modeling to link genotypes to phenotypes.[12][17][18]. PMC It is implemented both in eukaryotic and prokaryotic systems. 2001;30:87-104. doi: 10.1146/annurev.biophys.30.1.87. Helices B & D form direct contacts with the major groove of the loxP DNA. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). NAR 23, 3816-3821 (1995)) obtained from Dr. Saito I (Univ. Cre Recombinase is a Type I topoisomerase from bacteriophage P1 that catalyzes the site-specific recombination of DNA between loxP sites (1).The loxP recognition element is a 34 base pair (bp) sequence comprised of two 13 bp inverted repeats flanking an 8 bp spacer region which confers directionality (3). By expressing Cre at specific times or locations, you can precisely control expression of your gene of interest. There is a problem with the plasmid I received. Now, one point of crossover has occurred, which is what is called a Holliday Intermediate. The Cre enzyme targets loxP sites at the ends of this molecule and cyclises the genome. To deselect, click back on the plasmid. [29] The choice of Cre driver is also important as low expression of Cre recombinase tends to result in non-parallel recombination. In more recent years, however, this view has been challenged, and most of the current models for Int, Xer, and Flp recombination involve only limited branch migration (13 base pairs of the Holliday intermediate), coupled to an isomerisation event that is responsible for switching the strand cleavage specificity. "Site-specific recombination of DNA in eukaryotic cells", "Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae", "Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1", "Tissue- and site-specific DNA recombination in transgenic mice", "T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination", "Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting", "Subregion- and cell type-restricted gene knockout in mouse brain", "The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory", http://www.neuroscienceblueprint.nih.gov/factSheet/CreDriver.htm, "Skeletal and CNS defects in Presenilin-1-deficient mice", "Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces", "Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation", "Cyclization recombinase [Escherichia coli] - Protein - NCBI", "Targeted integration of DNA using mutant lox sites in embryonic stem cells", "A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination", "A genetic strategy for stochastic gene activation with regulated sparseness (STARS)", "Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications", "Non-parallel recombination limits Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation", "Genetically engineered mouse models in cancer research", "Spontaneous recombinase activity of Cre-ERT2 in vivo", "Epigenetic Regulation of Vascular Smooth Muscle Cells by Histone H3 Lysine 9 Dimethylation Attenuates Target Gene-Induction by Inflammatory Signaling", "Extensive Proliferation of a Subset of Differentiated, yet Plastic, Medial Vascular Smooth Muscle Cells Contributes to Neointimal Formation in Mouse Injury and Atherosclerosis Models", "Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury", "KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis", "Lineage tracing of cells involved in atherosclerosis", "Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels", Introduction to Cre-lox technology by the "Jackson Laboratory", https://en.wikipedia.org/w/index.php?title=Cre-Lox_recombination&oldid=1156566276, This page was last edited on 23 May 2023, at 14:02. Codon-improved Cre recombinase (iCre) expression in the mouse By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. Independently, Joe Z. Tsien has pioneered the use of Cre-loxP system for cell type- and region-specific gene manipulation in the adult brain where hundreds of distinct neuron types may exist and nearly all neurons in the adult brain are known to be post-mitotic. The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely. Expression of . Curr. Check out these options: Receive the latest news, hot plasmids, discounts and more. We found that in addition to complete loxP-mediated recombination, limiting levels of the Cre recombinase also cause incomplete recombination of the STOP cassette. CRY2-CreN and mCherry coexpression; Light inducible; Use with plasmid 26889. An additional level of control can be achieved by using his Cre recombinase, engineered to reversibly activate in the presence of the estrogen analogue 4-hydroxy tamoxifen. [6] A commonly used SSR system for this type of study is the Cre/lox P system, although other popular tyrosine recombinase systems include Flp, -integrase, and Dre. Please note: Your browser does not support the features used on Addgene's website. A separate recombinase recognizes and binds to each of two recombination sites on two different DNA molecules or within the same DNA strand. will appear in a new window. [1] The enzyme requires no additional cofactors (such as ATP) or accessory proteins for its function. [8] This is done through the fusion of a mutated ligand binding domain of the estrogen receptor to the Cre recombinase, resulting in Cre becoming specifically activated by tamoxifen. In vivo, the function of Cre recombinase is to circularize the P1 genome during infection and maintain the genome in the monomeric state for cell division. Common construct types include: Note: Through a partnership with genOway, Addgene is able to distribute materials containing FLEx technology. Open the file with The region between the two locations will be highlighted. 2004 Jan;6(1):7-28. doi: 10.1016/s1534-5807(03)00399-x. and include Addgene in the Materials and Methods of your future publications. Mutagenic. The exact sequence is given below; 'N' indicates bases which may vary, and lowercase letters indicate bases that have been mutated from the wild-type. Fields, Pathways This dimer then binds to a dimer on another lox site to form a tetramer. How do I prepare and deposit my plasmids? Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. This provides the advantage that the Cre recombinase is active for a short time. By continuing to use this site, you agree to the use of cookies. DNA between directly repeated loxP sites will be excised in circular form while DNA between opposing loxP sites will be inverted with respect to external sequences. It is widely used for knockout and knockin studies: the DNA sequence located between two tandem repeats of loxP sites can be excised to create a deletion mutant, or a plasmid containing a single loxP site can undergo site-specific integration into a . 0.3 mg/ml BSA Depending on the orientation of the loxP sites, deletions, inversions, or chromosomal translocations are generated. SnapGene software or the Please note: Your browser does not support the features used on Addgene's website. Cre activates gene of interest, using 4 mutated lox sites, 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, Multibow uses a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination to maximize label diversity, Rosa26 targeting vector, Cre dependent tdtomato expression. In addition to this Cre is also used to resolve dimeric lysogenic P1 DNA that forms during the cell division of the phage.[7]. SnapGene File: Plasmid sequence and SnapGene enhanced annotations. Search the sequencing result sequence against a nucleotide database: Translate the sequencing result sequence and search against a protein database: Enter in a sequence to align against this plasmid sequencing result sequence: Displays a graphical map based on nucleotide sequence data labeled with restriction enzymes, Please note: Your browser does not support the features used on Addgene's website. Editing, Cloning An important application of the Cre-lox system is excision of selectable markers in gene replacement. What is the molecular weight of Cre Recombinase? pHet-Act1-2. If you run into any problems registering, depositing, or ordering please contact us at [emailprotected] (n.b A Histidine occupies this site in other tyrosine recombinase family members and performs the same function). Filter on number of cut sites or search by enzyme name. The enzyme's unique and specific recombination system is exploited to manipulate genes and chromosomes in a huge range of research, such as gene knock out or knock in studies. There is a problem with the plasmid I received. Open the file with a text The P1 phage is a temperate phage that causes either a lysogenic or lytic cycle when it infects a bacterium. 2022 Oct 13;22(20):7763. doi: 10.3390/s22207763. 38 kDa Cre recombinase recognizes the loxP sites of specific 34 bp DNA sequences. pcDNA6 myc-His A. pCMV6-AC-YFP. This system can be implemented without inserting any extra supporting proteins or sequences. What do I need to know about the customs and importation process for my country? Therefore, researchers often use transgenic mice expressing CreERt2 recombinase induced by tamoxifen administration, under the control of a promoter of a gene that marks the specific cell type of interest, with a Cre-dependent fluorescent protein reporter. During genetic recombination, a Holliday junction is formed between the two strands of DNA and a double-stranded break in a DNA molecule leaves a 3OH end exposed. & ORFs. Products and content are covered by one or more patents, Please enter a quantity for at least one size, DNA Modifying Enzymes & Cloning Technologies, Next Generation Sequencing Library Preparation, DNA Assembly, Cloning and Mutagenesis Kits, Supporting Infectious Disease Research & Development, DNA Modifying Enzymes and Cloning Technologies. Kozak consensus sequence was added before the start ATG.Myc-tag was added at the C-terminus of Cre. What do I need to know about the customs and importation process for my country? Genome Both 13bp repeat sequences on a single loxP site are recognized and bound by a Cre protein, forming a dimer. Fields, Pathways Expressed in chondrocytes, mCherry and Cre-ERT2 - Tamoxifen inducible; Targeting vector for Pax7, Venus and Cre-ERT2 with MCS for inserting promoter, Emerald and Cre-ERT2 with MCS for inserting promoter, TFP and Cre-ERT2 with MCS for inserting promoter, tdTomato and Cre-ERT2 with MCS for inserting promoter, Venus, Cre-ERT2, targeting vector with MCS for homology arms, Emerald, Cre-ERT2, targeting vector with MCS for homology arms, TFP, Cre-ERT2, targeting vector with MCS for homology arms, tdTomato, Cre-ERT2, targeting vector with MCS for homology arms, GAG-Cre fusion; for the production of VLPs loaded with CRE protein, sfGFP-iCre-ERT2 (PAPGSTM N-terminus, unskippable linker) - Tamoxifen inducible, sfGFP-iCre-ERT2 (PAPGSTM N-terminus, GSAx9 linker) - Tamoxifen inducible, iCre-ERT2 (PAPGSTMA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PV N-terminus) - Tamoxifen inducible, iCre-ERT2 (PVV N-terminus) - Tamoxifen inducible, iCre-ERT2 (PVGSA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PA N-terminus) - Tamoxifen inducible, CreLite - Red light-inducible Cre; N terminus Cre fused to PIF6, CreLite - Red light-inducible Cre; C terminus Cre fused to PhyBdelta, CreLite system components, PhyBdeltaCreC and PIF6CreN, in middle entry vector (Tol2 kit), CreLite; Tol2 destination vector with mTagBFP2, CreLite system components, PhyBdeltaCreC and PIF6CreN, in AAV donor/transfer vector, CreLite system components, PhyBdeltaCreC and PIF6CreN, in lentiviral vector, Cre recombinase split with Vivid photodimers, Cre-ERT2 - Tamoxifen inducible; AAV donor vector, iCre-ERT2 (PAA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PAAA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PAAAA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PAGSA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PAAGSA N-terminus) - Tamoxifen inducible, iCre-ERT2 (PAGSAS N-terminus) - Tamoxifen inducible, N-terminal Cre component split with Vivid photodimers, C-terminal Cre component split with Vivid photodimers, Split Cre fused to nuclear localized wild-type VVD, Retinal ganglion cell-specific expression of Cre, Cre activates shRNA expression, removal of EGFP; See also similar plasmids, Cre turns off shRNA expression, removal of EGFP; See also similar plasmids, Cre activates gene of interest by removing Stop sequences; Puro selection. Addgene's viral service provides high-quality and high-titer viral preparations of plasmids that are currently in our repository. Further cleavage of Holliday Intermediates results in formation of Hybrid DNA. Its role is to maintain the phage genome as a monomeric unit-copy plasmid in the lysogenic state. Subsequent recombination of loxP sequences leads to rearrangements of the genome. Inducible Cre activation is achieved using CreER (estrogen receptor) variant, which is only activated after delivery of tamoxifen. [2] Early studies also demonstrated that Cre binds to non specific DNA sequences whilst having a 20 fold higher affinity for loxP sequences and results of early DNA footprinting studies also suggested that Cre molecules bind loxP sites as dimers. The Cre enzyme and the original Lox site called the LoxP sequence are derived from bacteriophage P1. from reference sequences and/or Sanger results (Addgene Assembled Sequence). PyMT mice were crossed with Rosa26 LSL-MYC mice harbouring a human MYC allele preceded by a LoxP site-flanked translational stop sequence in the Rosa26 locus, and further onto the S100a8-cre . The loxP mutants are created to avoid these problems.[23]. Placing Lox sequences appropriately allows genes to be activated, repressed, or exchanged for other genes. By continuing to use this site, you agree to the use of cookies. LoxP consists of a central 8-bp asymmetric sequence flanked by two identical 13-bp inverted repeats. Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. We also offer solutions for automation, site-directed mutagenesis, as well as your favorite restriction enzyme, ligase or competent cell products. Multiple variants of loxP,[42] in particular lox2272 and loxN, have been used by researchers with the combination of different Cre actions (transient or constitutive) to create a "Brainbow" system that allows multi-colouring of mice's brain with four fluorescent proteins. The 3OH end is elongated (that is, bases are added) with the help of DNA Polymerase. You have been idle for more than 20 minutes, for your security you have been logged out. Please review and update your order accordingly If you have any questions, please contact Customer Service at freezers@neb.com or 1-800-632-5227 x 8. Regulation of its activity has been accomplished to impart spatial and/or temporal control over recombination of the target gene. In genetics, floxing refers to the sandwiching of a DNA sequence (which is then said to be floxed) between two lox P sites. Cre activates gene of interest, removes Neo cassette. What strain of bacteria does my stab contain? This process occurs in the active site of two out of the four recombinase subunits present at the synapse tetramer. Dev Cell. Upon infection of a cell the Cre-loxP system is used to cause circularization of the P1 DNA. The carboxy terminal domain of the enzyme consists of amino acids 132341 and it harbours the active site of the enzyme. The P1 plasmid is relatively large (90Kbp) and hence exists in a low copy number - usually one per cell.