...(3xFLAG, Myc, V5, or HA). Tag-specific PCR genotyping can tell if HDR completely failed. If C-terminus...Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, Gogolok S et al. 2017. ...pitfalls: they require construction of plasmid vectors and/or rely on cumbersome in vitro synthesis of...purchased and there is no need to construct plasmid vectors or synthesize/purify IVT sgRNA in the lab. Figure... (although we could successfully knock-in non-expressed genes with similar efficiency, Dewari et al. 2018...tagging success, although many of these genes were expressed at really low levels so actual knock-in success...available for human and mouse genomes only and deals with C-terminus epitope tagging. If you work with other model...

...transfected with both vectors. Flex vectors are the solution. Flex vectors expressing fluorescent cytoplasmic...concentrations of vectors expressing the required recombinase. The high concentration of FLEx vectors guarantees.... It became possible to design FLEx vectors (Flip-excision vectors) that only allow one inversion event...types in neuronal networks Double inverted vectors expressing light inducible ion channels (plasmid # 20298...cell specific manipulations in vivo, FLEx vectors expressing the light activatable ion channel under the...optimum of 30 °C and is therefore inefficient in mammalian cells (usually grown at 37 °C). Smart molecular...specific) control of DNA recombination and gene expression can be advanced by the coordinated use of fellow...

...luciferase, cre/lox, yeast expression, worm expression, insect expression, plant expression, mouse targeting, ...biosensor affinity for calcium.”  Primary Vector Type The vector type should describe how your plasmid ...to be used. Choose from: mammalian expression, bacterial expression, lentiviral, retroviral, AAV, RNAi...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...difficult to grow. Growth Temp Choose from 30 °C, 37 °C, or room temperature. Growth Strain Please...plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids ...frog), D. rerio (zebrafish), D. melanogaster (fly), C. elegans (nematode), S. cerevisiae (budding yeast)...

...About Viral Vector Elements Learn How to Do Genome-Wide Screens with CRISPR Lentiviral Vectors Additional...becomes cloudy. Mycoplasma compete with host cells for nutrients and can alter expression of receptors, ..., Margison G.P. “Mycoplasma infection significantly alters microarray gene expression profiles.” Biotechniques...often employed to establish cell lines that stably express a gene of interest. While the general approach ...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...on a variety of factors such as the transgene expressed, the viral storage buffers used, and other experimental... by the envelop protein. Many times lentiviral vectors are engineered with non-native envelopes, a process...

...This is a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA in...this is a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA of...the top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang...Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone. This...mammalian cell types. Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter...N-terminus. This tag can be used to test for SpCas9 expression post transfection by western blot. Overall, this...mammalian cell lines. Alternative forms of this vector contain GFP-tagged and un-tagged versions of SpCas9...

...Yeast Expression Vectors Find Bacterial Two Hybrid Plasmids Read our Blog Post About Yeast Vectors Read...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent...activation. Vary the expression levels of bait and prey proteins - overexpression can lead to false interactions...During this period, proteins were successfully expressed as fusions that retain their individual activities...see Figure 2a) (4). These fusions are then co-expressed in a yeast strain that also contains a reporter...bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s interact, this...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...

...sequence into distinct expression vectors using the enzyme BbsI. These expression vectors each contain Type...Lentiviral Expression Systems (MuLE) Kit: This kit can be used to create lentiviral vectors expressing wt humanized...containing vector using Golden Gate assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for ...for ordered assembly into a destination vector that expresses Cas9. This is illustrated in the schematic...Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt Cas9, dCas9...Each destination vector contains GFP, enabling you to select cells with high GFP expression. These cells ...enables users to express up to 7 gRNAs. The kit contains different destination vectors depending on the...

...to reduce basal expression levels; expression vector cannot have p15A origin of replication F- ompT lon...) pLysE has higher T7 lysozyme expression than pLysS; expression vector cannot have p15A origin of replication...Rhamnose-tunable T7 RNAP expression alleviates inclusion body formation. Expression vector cannot have p15A origin...for recombinant protein expression. Many challenges can arise when over-expressing a foreign protein in ...to avoid them. Why do I need an expression strain? Protein expression from high-copy number plasmids and...Addgene's curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains ...General protein expression BL21 (DE3) pLysS* Chloramphenicol (pLysS) pLysS expresses T7 lysozyme to ...

... A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding ... offers vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2...SARS-CoV-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia ...voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator, created...administering these AAVs with a dilute Cre-expressing vector resulted in a sparsely-labeled field of view...high microglia-specific gene expression when incorporated in an AAV vector (Figure 5). Efficiency and specificity...of miR124T was inserted into the vector to silence transgene expression in neurons and other non-microglia...

...spatiotemporal expression of a gene of interest. This type of control over gene expression is possible in...created a set of modular vectors that can be used to optimally control gene expression in specific cells of...are interested in C. elegans plasmids and resources check out Addgene’s Worm Expression page. Looking for...an ortholog in C. elegans making it ideal to study basic molecular biology processes. C. elegans are also...PMCID: PMC5525645. Mello, Craig C., et al. "Efficient gene transfer in C. elegans: extrachromosomal maintenance...Wei, et al. "From Saccharomyces cerevisiae to human: The important gene co‑expression modules." Biomedical... al., 2009). By injecting mice with retroviral vectors that harbored different mutations found in leukemia...

...-baToN, “sender” cells express GFP on the surface and “receiver” cells express an anti-GFP nanobody on...response 1 (TIR1) expressed under a desired promoter with a specific spatiotemporal expression pattern and ... nanobody cross-reacts with BFP, sender cells expressing surface BFP generated BFP-labeled receiver cells...visualized.  The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap cloning systems...for delivering CRISPRi components by a single AAV vector are now available from Tonia Rex's lab.  vfCRISPR...Jacobs-Wagner’s lab produce different levels of dCas9 expression and contain different antibiotic resistance markers...from Hongkui Zeng's lab are available for dual expression of cytoplasmic-tdTomato and Synaptophysin-GFP...

... existing transposon and vector systems and creating an all-synthetic vector that included only the elements... into a destination vector in a predefined order. Finally, the destination vectors are used with Multisite... eight entry vectors can be used for each Multisite Gateway compatible destination vector for a maximum... to Addgene, including lentiviral and AAV expression vectors for certain variants. Klapoetke et al.,...step, compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional...Cre-based Brainbow-expressing cells. Autobow plasmids are all-in-one versions which express self-excising ... May 5;10(6):540-7. pCoofy vectors for optimizing protein expression Under the direction of Sabine ...

...to be expressed. These permit exquisite control of gene expression and tracing of gene expression across... basal expression. Co-expression of repressor QS along with QF leads to intermediate expression from QUAS...transgene permits directed expression of that gene in cells that also express GAL4. Usage of this system...UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with...basal state, and co-expression of the inducer QF can increase QUAS driven expression by ~3,300-fold in ... Shen. “Controlling gene expression with the Q repressible binary expression system in Caenorhabditis ...SO, Schmidt-Dannert C. Development of a synthetic cumate-inducible gene expression system for Bacillus...

... Creation of C-terminal Firefly luciferase fusion for expression in plants, co-expressed with mScarlet-I...Gaussia Creation of C-terminal Red Luciferase fusion for expression in plants, co-expressed with Gaussia luciferase...Multi-luciferase reporter vector including transcriptional reporters for NF-kb, TGF-b, c-Myc, p53 and MAPK/JNK...regions Joshua Mendell 212936 pGL3 Basic Vector Firefly Vector for investigating regions controlling transcription...as a reference level of expression. Use this table to find plasmids expressing luciferase. We also offer...Retroviral expression of firefly luciferase Scott Lowe 14080 pAKlux2 Lux Operon Bacterial expression of luciferase...Mammalian expression of Nano-lantern Takeharu Nagai 87121 pcDNA-RLuc8 Renilla CMV Mammalian expression renilla...

...to create recombinant vectors that can replicate in different bacteria and express proteins within different... S. (2011). Broad-host-range plasmid vectors for gene expression in bacteria. Methods in Molecular Biology...Gill, R. T. (2010). Broad-host-range vectors for protein expression across gram negative hosts. Biotechnology...Cloning Vectors for Gram-Negative Bacteria. In R. L. Rodriguez & D. T. Denhardt (Eds.), Vectors (pp. 287... inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid host range...features to create broad host range recombinant vectors.  A plasmid’s host range is determined by the ...groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids are...

...modular vector assembly process designed for a quick, plug-and-play approach to cloning CRISPR vectors (McGee...for each module, generate the vector file, and quickly clone their vector of interest. And it truly is ...destination vectors for various delivery methods, along with compatible inserts. All destination vectors contain...heavy lifting out of CRISPR vector design, making it easier to get the vectors that are just right for your...Fragmid destination vectors currently available. Table 1: Fragmid destination vectors Organism Delivery...designing a CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid...plasmid and generating the vector file on the Fragmid website.   If the vector is to your liking, your...

...variety of pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low...easier to produce the constructs you need. Expression vectors within this kit contain a variety of promoters... oocytes. The expression vectors additionally contain various combinations of protein tags (His, Avi, ... with the problem of creating vectors for predictable protein expression in bacteria, researchers at the...Process with pCri Cell-free protein expression kit Cell-free expression systems have been improved for both... the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of proteins in ...comparison of different cell-free expression systems; the Cell-free expression test kit represents a set of...

...Addgene  Choosing an expression system Choosing an appropriate expression vector is the first step in .... (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs ...are binary gateway vectors and can be used for promoter swapping and generating N and C terminally tagged...for visual selection of transformants based on expression of RFP in the seed (Emami, Yee and Dinneny, 2013...many insertions can cause artifacts from over-expression and it is likely that such a construct will be... ‘A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology’, Frontiers in Plant...sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food...

...codon optimized specifically for expression in yeast. These tagging vectors are based on previously described...interest. These fluorescently tagged genes are expressed under native conditions and allow scientists to...described pFA6a-link vectors and include a Kan, SpHIS5, or CaURA3 selection marker. Lee et al.(1) assessed...from these yeast-optimized fluorophore tagging vectors for your single or multi-color imaging experiments...gene deletion, overexpression (by promoter substitution), and tagging at either the N- or C-terminus (3xHA...artifacts sometimes associated with plasmid-based overexpression. Tim Formosa, at the University of Utah, has...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

...Assay for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral...marker. Determining the titer of your lentiviral vector allows you to control the multiplicity of infection...protocol was developed using low titer lentiviral vectors (10 5 TU/mL). If you anticipate that your viral... transduction, cells were assayed for mCherry expression using fluorescence microscopy. Last reviewed ...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...obtaining accurate titers. Check the cells for mycoplasma (Link opens in a new window) regularly Do not...