...typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive cells, or selection...transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify and enrich positive...repair template and displays reduced efficiency compared to NHEJ knockout. Base editors can make a limited...our AAV Guide ) ∼4.5 kb packaging limit (only compatible with smaller Cas enzymes) Transient or stable...Cas9 and gRNA Choose a delivery method that is compatible with your experimental system. CRISPR efficiency...

...another marker, such as GFP. A researcher can use FACS to sort cells expressing GFP and later expand these...restriction sites but compatible with the Gateway® cloning system? Gateway® compatible vectors use recombination...generation or 3rd generation packaging system. For a comparison of the key differences between the 2nd and 3rd...lentiviral preference for transcribed genes. Studies comparing the lentivirus HIV and the retrovirus MMLV indicate...using this site include subcloning or appending compatible restriction sites onto the insert of interest...until the gene of interest is flanked by sites compatible with those in the vector into which one ultimately...

...measurable through an epitope tag such as FLAG/His/GFP. Fluorescent protein tags, which correlate closely...intracellular labeling because the intracellular compartment may contain cross-reacting antigens not present...difference in the efficacy of your antibody. When compared, 4% PFA and 0.1% saponin; 4% PFA and 0.1% Triton...intensity in the KO population (pink histogram) compared to the WT cells (green histogram). The unfilled..., like RNAseq or proteomics, it is possible to compare antibody labeling of different cell types within...expression of the target. In this case, you could compare the antibody’s labeling of the cell lines to the...

...cassette comprised of OCT4, SOX2, LIN28, NANOG, and a GFP reporter. Minicircles are removed from cells by dilution...efficiency of reprogramming when compared to other methods, even when compared to integrating viral approaches...heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing of transgenes...and have a higher efficiency of generating iPSCs compared to other delivery methods. Retrovirus has a cloning...lentivirus has similar rates of reprogramming when compared to MMLV-derived retroviruses, it is not as well...heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene expression...Non-integrating methods have a smaller genetic footprint compared to integrating approaches. These methods eliminate...

...-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked... Listen to Our Tol2 Gateway-Compatible Toolbox Podcast Segment The impact of KLF4 N-terminal variants ...lead to comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different...along with EGFP while retaining an emission spectrum that is easily separable from that of EGFP. This makes...latest hot plasmid updates!     Tol2 Gateway-compatiable toolbox for studying the nervous system The... Cole lab recently deposited the Tol2 Gateway-Compatible Toolobox, which will allow scientists to quickly... neuorodgenerative diseases. The Tol2 Gateway-Compatible Toolbox is based on the original Tol2kit generated...

...plasmids allow cloning of protein coding sequences with GFP-LOVpep, cpPDZ, ePDZb and ePDZb1 as tags. pDawn/pDusk...H134 Widely used variant with larger photocurrent compared to CrChR2 450 ChETA E123T mutation; creates faster...applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat Methods. 9(...

...fluorescent protein (EGFP) reconstitution. (a) Scheme of validation for DSB mediated EGFP expression cassette...halves of EGFP to recombine by homology directed repair, and resulting in the expression of EGFP. Using ...ordering the sgRNA is simple and easy. Several companies supply gRNA sequences, such as GenScript, ThermoFisher...order them yourself through whatever DNA supply company you currently use. You can also find many previously...strand annealing) took place and reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP target plasmid...pCAG-EGxxFP target plasmid contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter. The...can be placed in multi-cloning site (MCS) between EGFP fragments. The pX330 plasmid contains humanized ...

...fluorescent marker like green fluorescent protein (GFP), creating a customizable DNA or RNA label for fluorescence...nuclease pair for every genomic target. Due to its comparative simplicity and adaptability, CRISPR rapidly became...it meets two conditions: The sequence is unique compared to the rest of the genome The target is present...throughout the genome, called off-targets, that can impact your experiment. There are many online tools available...effects Sniper-Cas9 - less off-target activity; compatible with truncated gRNAs to increase specificity ... offer high efficiency and low indel formation compared to HDR. Prime editing requires more optimization...) includes tnsA , tnsB , tnsC , and tniQ . In comparison, type V-K includes just tnsB , tnsC , and tniQ...

.... All vectors include the germline expression compatible tbb-2 3’UTR. For both the eft-3 and hsp-16.48...generated variants with and without a C-terminal EGFP fusion. For the sgRNA, vector pMB60 allows in vitro...47944 pMB62 : Expresses C. elegans optimized Cas9::mEGFP from the eft-3 (eef-1A.1) promoter. 47945 pMB63 ...47946 pMB66 : Expresses C. elegans optimized Cas9::mEGFP from the hap-16.48 promoter. 47947 pMB67 : Expresses...

...biological properties of new fusions can be compared with mEGFP or mTurquoise2, which are established true...fluorescent protein. In the jellyfish Aequorea victoria, AvGFP forms a homodimer. In corals, the red fluorescent...detect weak dimerization tendency, as is present in EGFP. Constantini et al, (2012) developed a cell-based...fluorescent protein is monomeric without the accompanying data to prove it is worthless. The OSER assay...that you will be using. If you do a head-to-head comparison right, it will be valuable information for the... are engineered can be shared to simplify the comparison by others. Together, we can build a valuable ...

...protein they tested — E. coli expressing good ol’ EGFP — showed a slight but detectable change in fluorescence...better, multiple other labs have reproduced their EGFP findings since the initial report in 2023.)   ...   Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response to a ~25 mT...proteins display a similar effect (many do). Fusing EGFP to the self-labeling FlavinTag made a reliable one-component... the proteins that had shown magnetic responses: EGFP, mScarlet, and AsLOV2. After several rounds of semi-random... semi-random mutagenesis and screening, the EGFP and mScarlet variants were showing no obvious signs of...-up studies. “We don’t have a vision of passive impact,” he says, noting that his team regularly shares...

...enhancer regions (Mendenhall et al., 2013). By comparing gene activation when enhancers were active or ...increased gene activation at the targeted site compared to other Tet1 delivery systems. Transcriptional...) Vlatka Zoldoš’ lab has deposited pdCas9-DNMT3A-EGFP and pdCas9-DNMT3A-PuroR for targeted cytosine methylation...methylation in mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced...Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase...epigenome-editing tools. pLV hUbC-dCas9-MQ1(Q147L)-EGFP is also available for lentiviral transduction. Lysine-specific...

...including Myc, HA, Flag, GST and eGFP epitopes. Zebrafish Tol2 Gateway-compatible Kit for Neurodegenerative ...Sauka-Spengler Lab. A genetic binary system for cell- and compartment-specific biotin labeling in zebrafish. pGCS Vector...

...proteins, which also serve effector roles). A compatible GEF, GTPase, and GAP set is referred to as a ...Arap2 116984 1704 GAP Arap3 64411 1544 Mammalian (pEGFP-C2) GAP Acap1 9744 740 Mammalian (pFLAG-CMV2) GAP... 9267 398 Gateway GEF Cyth2 9266 400 Mammalian (pEGFP-C1), Gateway GEF Cyth3 9265 399 Gateway GEF Cyth4...

..., and disease. In order to expand their model’s impact, organoid users have recently begun to develop ...circularly permuted green fluorescent protein or cpGFP) to the petunia SL enzymatic receptor DAD2. SLs ...which modulates the fluorescence intensity of the cpGFP without altering that of the internal control (Fig...these novel biosensors could have a major economic impact.   Design and mechanism of action of the...

...Human Genes TALENs for the EGFP Reporter Gene Additional Plasmids compatible with the REAL Assembly TALEN...

...photobleaching, and achieve better signal in vivo as compared to imaging with broccoli aptamers or MS2 tags ...real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry fluorescence...involves tracking the ratio of mCherry-positive to eGFP positive cells through fluorescence microscopy or...

...Delivering gRNA and Cas9 using adenoviral vectors As compared to other viral vectors, AdVs can provide a large... pAdSh.PGK.Cas9, pAdSh.U6.gRNAS1, and pAdSh.U6.gRNAGFP. Gonçalves says that advantages of AdVs include...guide RNA constructs, pAdSh.U6.gRNAS1 and pAdSh.U6.gRNAGFP, and also Adeno Cas9 and Adeno EA. Or if you're...

...fluorescent proteins, how to do a head-to-head comparison of fluorescent proteins and how to make a well-informed...your selection on a small-scale head-to-head comparative study under conditions that best mimic the intended... QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number, the higher the theoretical...protein properties and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (...is of no use. Therefore, it is more sensible to compare fluorescent proteins based on their practical brightness...promising fluorescent proteins and to determine and compare their practical brightness in a system closely ...application. In figure 2 we provide an example of the comparison of the practical brightness of two orange fluorescent...

...Protein Property Visualization allows for visual comparison of FPs and how they relate to one another. .  ...FPbase Fluorescence Spectra Viewer can be used to compare excitation and emission spectra when available,...relating to that specific protein (for instance: EGFP).  Users can “favorite” certain proteins, or create...you or your lab use frequently) for the sake of comparison or ease of retrieval.  These collection pages...store and represent within-study photobleaching comparisons.  We very much appreciate feedback, so try out...