...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...

...Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone. This...top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang Lab.... TLDR: This is a versatile mammalian expression vector that you can use to express both SpCas9 and your...Overall, this is a versatile mammalian expression vector that you can use to express both SpCas9 and your...mammalian cell lines. Alternative forms of this vector contain GFP-tagged and un-tagged versions of SpCas9...site). Finally, in addition to the 2A peptide on its C-terminus, SpCas9 is also fused to a 3X FLAG tag at...SpCas9 is fused to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables...

...genome engineering methods for C. elegans were released, signaling a new era in C. elegans research. Homology... arms for your gene of interest into the FP-SEC vector using Gibson assembly. This construct will serve...of custom genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification...the University of North Carolina has made CRISPR in C. elegans even easier - now, one can generate a fluorescent...-based gene tagging approach Multiple features of C. elegans have made CRISPR/Cas9 so successful in this...previously used a transgene selection strategy for their C. elegans/CRISPR experiments, but this strategy required... make it possible fluorescently label proteins in C. elegans, they inserted the SEC into a synthetic intron...

... A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding ...SARS-CoV-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia ...incorporated in an AAV vector (Figure 5). Efficiency and specificity of these AAV vectors for microglia was...human cells. Addgene offers vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression...microglia The REVeRT Dual AAV Vector System Neurodegeneration Collection updates   Multiplexed perturbation ...Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator...and Voltron2 are available as Cre-dependent AAV1 vectors and are suitable for extended imaging of neurons...

...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent...Expression Vectors Find Bacterial Two Hybrid Plasmids Read our Blog Post About Yeast Vectors Read our Blog... is to use a number of different bait and prey vectors. This has been shown to be as effective as using...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the ... prey libraries can be screened using both N- and C-terminal fusions of these proteins. This way both ...Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively ...

...into distinct expression vectors using the enzyme BbsI. These expression vectors each contain Type IIS restriction...Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt Cas9, dCas9...create lentiviral vectors expressing wt humanized Cas9 and up to three gRNAs. Entry vectors containing the...wt hCas9 entry vectors are supplied with the kit, you can clone your own entry vectors containing other...containing vector using Golden Gate assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for ...arrays (e.g. for spacers A, B, and C, you could obtain arrays A, B, C, AB, AC, BC, and ABC). Nielsen Lab...assembly of multiple components into a destination vector. The first step in CRISPR/Cas9 Golden Gate multiplexing...

...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... al., 2009). By injecting mice with retroviral vectors that harbored different mutations found in leukemia...Gerald Rubin’s lab who created a set of modular vectors that can be used to optimally control gene expression...lab to study neuronal development (Brenner, 1973). C. elegans are small, transparent worms that have a ...life cycle and large brood sizes. The majority of C. elegans are also self-fertile hermaphrodites making... Nobel Prize in 2006. One of  major advantages of C. elegans is that they are transparent throughout their...

...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... Gether lab, now available as ready-to-use AAV vectors.   ...interactions New auxin inducible degron system for C. elegans New CRISPR plasmids New items from the viral...tool-kit for the auxin inducible degron (AID) system in C. elegans By Alyssa Cecchetelli The auxin inducible... degron (AID) system has been extensively used in C. elegans since 2015 to conditionally degrade proteins...repression, and editing with a single Cas9-VPR in Saccharomyces cerevisiae. To control Cas9 activity and influence...

... pREP4*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter (found on vectors such as pQE ...lysozyme to reduce basal expression levels; expression vector cannot have p15A origin of replication F- ompT...higher T7 lysozyme expression than pLysS; expression vector cannot have p15A origin of replication F- ompT...codons not normally used in E. coli. Expression vector cannot have p15A origin of replication F- ompT ...alleviates inclusion body formation. Expression vector cannot have p15A origin of replication fhuA2 ...lac repressor on the pREP4 plasmid). Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15...conditions. Many recombinant proteins express better at 30°C or room-temperature, which is accomplished by growing...

... 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...step, compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional...of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers from Table...homology between the vector and gene primer for SLIC cloning. Amplify the pCoofy vector and the sequence ...recombination in SLIC cloning to form the desired vector. The pCoofy vectors contain the ccdB cassette, which is not...

...posts related to viral vector protocols and tips Read all of our viral vectors blog posts Resources on...calculate the viral titer: GC/mL = {[(R*C)(1000/V)]*D} R = Reaction volume C = Copies/uL V = Volume of virus ...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ...evaluation of recombinant adeno-associated viral vectors." Human Gene Therapy ja (2019). PubMed PMID: 31140327...single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR." Human gene...titration by qPCR Browse our collection of viral vector related protocols Learn more about the Addgene ...Song, Neng, et al. "Detection of circulating Mycobacterium tuberculosis-specific DNA by droplet digital...

...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...   Building broad host range recombinant vectors Replicons, which consist of a plasmid’s ori and...range plasmids can be used to create recombinant vectors that can replicate in different bacteria and express...incorporating broad host range replicons, recombinant vectors can be modified further to increase the range of...

...yeast. These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5...from these yeast-optimized fluorophore tagging vectors for your single or multi-color imaging experiments...substitution), and tagging at either the N- or C-terminus (3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe...

.... (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...expression system Choosing an appropriate expression vector is the first step in generating transgenic Arabidopsis... ‘A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology’, Frontiers in Plant... plant selection markers (basta, hygromycin, kananamycin, tunicamycin) (Nakagawa et al., 2007). Alternatively...

...and improved. Takara’s “2nd generation Tet-Off vectors (Tet-Advanced)” are the most recent variants of...Recent work has even used all 3 in a single modular vector for ease of use! Conclusion This post contains ... Drosophila Terminators and PolyA Signals FLEx Vectors Resources at Addgene.org Tetracycline Collection...also shown that the Q system is also functional in C. elegans. Subsequent work by the Luo and Potter labs...23995928; PMCID: PMC3811519.Seo SO, Schmidt-Dannert C. Development of a synthetic cumate-inducible gene ...; PMCID: PMC5270762. Wendler, F., Park, S., Hill, C. et al. A LexAop > UAS > QUAS trimeric plasmid to ...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...

...of pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance...to produce the constructs you need. Expression vectors within this kit contain a variety of promoters ...insects, and even Xenopus oocytes. The expression vectors additionally contain various combinations of protein...Series Confronted with the problem of creating vectors for predictable protein expression in bacteria,...this so-called bicistronic design (BCD). The 49 vectors in this kit can be used to drive production of ...Dejan Gagoski used SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-...kit represents a set of 88 clones in a pCellFree vector that enables protein expression in any in vitro...

...species into the pYES2.1/V5-His yeast overexpression vector. This plasmid contains origins of replication and...Resources on Addgene.org Find Yeast Expression Vectors Check Out Our Molecular Biology Reference Pages...procedure adds both V5 and His6 epitope tags to the C-termini of Met proteins, which can be used to confirm...methionine synthesis (5). The budding yeast, Saccharomyces. cerevisiae, makes a great reference organism... or not proteins from the fission yeast, Schizosaccharomyces pombe, have the same function as their counterparts...yeast for budding geneticists: A primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48...yeast for young geneticists: A primer on the Schizosaccharomyces pombe model system. Genetics 201, 402-423...

...BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP conversion and to target specific...expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome would restore...codon that is converted to a ‘TAC’ or ‘TAT’ after a C-to-T base editing event. This edit changes the histidine... ATG codon from an ACG codon using a base-editor (C>T conversion) they could induce the translation of...Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464... a mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image adapted from Katti ...

...Pseudomonas fluorescens, and Pseudomonas syringae. These vectors contain BsaI sites for easy cloning of a gRNA of... of cytidine and adenine base editors that enable C-to-T and A-to-G base substitutions respectively. Substitutions...:723–728 . https://doi.org/10.1021/sb500351f Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG (2018... https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen LE, Sommer MOA, Nielsen AT (2016) CRMAGE...Vridhambal GS, Rivera AJ, Montgomery SA, Fortier L-C, Barrangou R, Theriot CM, Ousterout DG (2020) In Vivo...shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces bacteria produce a wide...created two “pCRISPomyces” systems for use in Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes ...

...journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library for Advanced...kit has a focus on infrastructure with shuttle vectors, several delivery systems which can be engineered...engineered into the TUs, and even viral gene silencing vectors. The Plant Parts III kit has a focus on transformation...Gantner, Johannes, et al. “Peripheral Infrastructure Vectors and an Extended Set of Plant Parts for the Modular...Rapid Design and Production of Diverse Expression Vectors.” ACS Synthetic Biology, vol. 6, no. 7, American...acssynbio.7b00016. McGee, Abby V., et al. Modular Vector Assembly Enables Rapid Assessment of Emerging CRISPR...Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology, vol. 12,...