...cleaved and released. These constructs tend to be smaller than constructs with multiple promoter-gRNA ...to clone and transfect multiple promoter-gRNA constructs in order to target multiple sites! In this post...researchers to express multiple gRNA from a single construct. DNA and gRNA are not to scale.   Let’s start... restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent to the ...restriction sites that flank the promoter-gRNA constructs. When these plasmids are digested, unique overhangs... filler sequences. To build your multiplexing construct, you clone all but one of your gRNAs into spectinomycin-resistant...the Cas9-containing destination vector. These constructs are digested using BsaI and assembled to produce...

... piece of DNA designed in the lab. Specially constructed plasmids can be used to replace genes in yeast...random recombinants.   Design your targeting construct. For recombination to occur in a cell, a minimum...to 14 kb of homology is typical for targeting constructs. In the example shown in Figure 1, large sequences...gene. To avoid selecting for cells in which the construct has randomly integrated into the genome, a negative...or large portions of the plasmid. Deliver your construct to your target cells. After recombination, exon...insert and later remove a resistance gene. In this construct, the neomycin resistance gene is floxed so that...

...introduce your sgRNA sequence into the pTargetF construct and then introduce both plasmids into the cell...study of N-acetylglucosamine biosynthesis, we constructed a strain library with 1-11 copies of integrated... is faster. Using MUCICAT only needs 2 newly constructed plasmids (pDonor and pQCascade) with 4 rounds...complete the N-acetylglucosamine strain library construction in 8 days. To build an equivalent strain library...library, our pEcCas/pEcgRNA system would need to construct 13 plasmids with 14 rounds of transformation in...without using screening markers. Capability to construct a strain library with different gene dosages to...developing microbial genome editing tools for faster construction or reprogramming of cells to understand the ...

...T., Szawlowski, P., & Randall, R. E. (1992). Construction of solid matrix-antibody-antigen complexes containing...developed CRBNmidi (Kroupova et al., 2024). This construct contains the Lon and TBD domains, part of the ...Kroupova et al., 2024 under CC BY license. This construct can be easily expressed and purified from E. coli...Kroupova, A. et al. (2024). Design of a Cereblon construct for crystallographic and biophysical studies of...mScarlet-I3, Oliver Hobert’s lab recently generated new constructs specifically for C. elegans (Cao et al., 2024...introns to improve expression. When using these constructs to tag the GOLG-4 protein, mScarlet3 was 50% ...mScarlet), and the new mScarlet3 and mScarlet-I3 constructs. Scale bar for all panels: 20 µm. Image reused...

...Howarth lab created the MBPx-SpyCatcher construct. This construct is used to produce a maltose binding protein...However, the laborious construction of repair templates and guide RNA constructs, as well as the difficult... Human Kinase Domain Constructs Kit, a library of human kinase domain constructs developed through a large-scale... that results in the creation of many E. coli constructs with greatly varied levels of expression. The...can be used to easily create protein expression constructs fused with epitopes or fluorophores, generate...platform for generating both simple and complex DNA constructs Have you ever wished you could design a gene...overexpression experiment, generate the necessary constructs, and complete screening in just 3 days? Thanks...

... and sequences in each plasmid, none of these constructs have been functionally tested by Ginkgo Bioworks...page.  Bacterial expression plasmids For those constructs to be used in bacterial expression systems, Ginkgo...tags (StrepII, 3xFLAG, 6xHIS, MBP, Myc). These constructs include proteins like nonstructural proteins ...Yeast expression plasmids The yeast expression constructs contain protein coding genes, tags, truncations...expression vectors in late March. The first set of 383 constructs went online just three weeks following initial...

...started, here are a few key details. Our set of constructs comprises all SARS-CoV-2 viral proteins except...viral protease, Nsp5. Our set also includes a GFP construct as a control. For easier portability, all viral...projects should be fun and straight-forward. All constructs are tagged with a 2xStrep-tag for easy detection...correctly-sized proteins by Western Blot for 25 constructs, and confirmed expression via mass spec for two...Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC and will be available shortly...

...CMV-TO) expression, or a promoterless construct. Pre-constructed Entry vectors containing Cas9, EGFP, ...of inducible promoter constructs. Up to 34 building blocks were used to construct 20kb long inserts. The...att sites are supplied as individual reporter constructs or as the fully assembled pMemoryArray.   Yang...Biotechnol Lett. 2015. 37:383-389. Toolkit for constructing fusion proteins with customizable N- and C-terminal... to 5 different elements. Lentiviral vectors constructed with the MuLE toolbox have been used for a variety...cloning method that simplifies the process of constructing specialized fluorescent reporter plasmids with...(Val-Glu) between each module to permit easy construction of fusion proteins. These three enzymes were...

...research and its therapeutic potential. Note: constructs discussed are based on Adenovirus type 5. Three... cells can be broken down into 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM ...popular method for creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector ...generate the final recombinant adenoviral plasmid construct, please see Addgene’s Adenoviral Guide webpage...HEK293 cells are transfected with the AdV plasmid construct from step #1 and allowed to stay in culture for... Jager, Lorenz, et al. "A rapid protocol for construction and production of high-capacity adenoviral vectors...

...assembled into a final transgenesis construct. Plasmids constructed using Golden GATEway cloning have been...complexity of assembling recombination of transgenesis constructs? A new cloning system combining the advantages...simplifies the cloning process for complex DNA constructs, particular for those involving recombination...Multisite Gateway cloning to generate the final construct. Up to eight entry vectors can be used for ...methods, particularly for complex transgenesis constructs. Kirchmaier et al., PLOS One 2013 Oct 7;8(10...The Janovjak lab has deposited their 3 Opto-RTK constructs – Opto-mFGFR1, Opto-hEGFR, and Opto-hRET – as...as well as various LOV-domain-mVenus constructs used in the study for those who would like to extend this...

...efficiency of Cre recombination varies for different constructs and cell types. Second, Cre may induce recombination... Cre itself, without the presence of a floxed construct, may produce a phenotype. This problem is especially...B3 (from Zygosaccharomyces bisporus.) Reporter constructs equivalent to site-STOP-site-myrRFP, with “site...these recombinases, each recombinase and reporter construct pair were tested for excision efficiency. B2 and...

...A newly established all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering...Yamamoto describe in their recent study, the construction system offers ease and efficiency for all-in-one...overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome engineering. Image...

...research and many different constructs are available to the community. Each construct has unique features, such...RNA (hpRNA). This method is facilitated by DNA constructs that enable insertion of ~400 bp complementary...sequence (discussed below). Examples of these constructs include pGAPi, which allows easy creation of ...reporter. This is achieved at the level of the DNA construct, where your RNAi-target is fused to the DNA backbone...phenotypes quantified. Fluorescent reporter-based RNAi constructs have been designed and widely implemented in ... gene silencing. We achieved this by creating constructs that directly fused any gene targeting sequence...are now available on Addgene. These include the construct that allows for easy insertion of any target sequence...

...assembly tool used to build single- and multi-gene constructs for a variety of applications. MoClo tools can...gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits and metabolic pathways...MoClo is commonly used to design TUs and multi-TU constructs for synthetic biology applications in bacteria...individual TUs or very large (up to 20!) multi-TU constructs (Moore et al., 2016). The variety of constitutive...multiple unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This allows for...is geared towards cloning multi-TU expression constructs (Martella et al., 2017). If you can dream up ...DNA elements on the behavior of an expression construct. Once you’ve identified your experimental requirements...

...floral dip). In the next generation the CRISPR construct needs to be segregated away to prevent continued... the naked eye in plants. When expressed, the construct produces 3 enzymes from a single mRNA using 2a...desired mutation but lacking both RUBY and the construct that rescues the mutation. I will be able to easily..., then tracking the segregation of the CRISPR construct could be achieved much easier than by PCR or a...

...elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform...Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage. Summary of...generally more efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified...duplicated. Validating your mutagenized plasmids Once constructs containing the desired modification have been... interfere with the function of the resulting construct. In cases where the functionally important regions...want to introduce a point mutation in a large construct. Particularly, if a pair of unique restriction...

...Bacterial Expression Construct SpCas9 Inhibition (E. coli) Mammalian Expression Construct SpCas9 Inhibition...expression to this cell type. To this end, they constructed AAV plasmids bearing one of the distal-less homeobox... in single dendritic spines. The set of CyRFP constructs available at Addgene includes bacterial and mammalian...mammalian mCyRFP1 or CyRFP1 expression plasmids, FRET constructs, and CaMKII and RhoA sensors. Laviv, et al. ...fusions. The Gadella lab has deposited all mScarlet constructs with Addgene. Bindels DS, et al. Nat. Methods...CRISPRi Essential gene Knockdown library was constructed to allow for systematic examination of essential...domain and transcription activation domain fusion constructs. This is the first deposit from the Ptashne lab...

...targeting system Heat-inducible lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder... 36690762.   Heat-inducible lentiviral CAR constructs by: Brian O'Neill  A clever system for reducing...system is a set of heat-inducible lentiviral CAR constructs, the most basic of which is depicted here: ...days - after just 15 minutes of FUS.  Similar constructs to the one above can be made for expressing your...

...shrink the construct. They identified ADAR2DD(delta984-1090), which decreases the REPAIRv1 construct size from... A->G editing, they employed hyperactive ADAR constructs, like ADAR2DD(E488Q). They also adjusted the ...binding to decrease off-targets. Of the tested constructs, ADAR2DD(E488Q/T375G) (REPAIRv2) had the highest...dRanCas13b truncations that would allow packaging the construct for viral delivery. They found C-terminal truncations...

...deliver a genetic cargo, typically an expression construct, to cells/organisms. This is often cDNA, shRNA...), it’s still used to deliver gene expression constructs. Sandwiched between two inverted terminal repeats...just like AAV, which encompass the expression construct and promoter that you wish to deliver to your ...plasmids in our collection. Want to make a vector construct on your own? No problem, we are here to help with...