... protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of a stop...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary...necessary for knock-in: one on either side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies...microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus (see figure below). The double-MMEJ...overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA...’ and 3’ microhomologies are added to the GFP-Puro cassette via PCR, and this construct is inserted into...their system in HEK293 cells, integrating the GFP-Puro cassette into the FBL locus. Upon sequencing of puromycin...

...GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives GFP and ...to bind GFP. The idea of using GFP as a scaffold suddenly seemed very realistic. Envisioning GFP as a substrate...presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted...selectively manipulate only GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies...tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP. Once they’d found...culture system. In cells cotransfected with GFP and the GBPs, GFP linked the two GBPs together to create a...

... switch conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA...For this FLEx switch to successfully work, the cassette would need to contain the BFP coding sequence ...antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by two pairs of target sites-...DNA sequence (such as GFP) in the sense orientation, followed by coding sequence of the gene you would...in the opposing orientation, you have created a cassette that will express GeneR only in the presence of...recombination event would excise the reporter from the cassette, allowing for expression of GeneR. This allows...et al. Nat Biotech 2003): In the presence of Cre, GFP expression is lost, allowing for rescued expression...

...array plus a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted...Lentiviral Expression Cassettes lentiviral transduction One vector expresses Cas9, eGFP, and up to 4 gRNAs... own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection gRNAs expressed from a polycistronic...co-transfection of Cas9 Nissim et alTsai et al PTG Cassettes transfection gRNAs expressed from a polycistronic...4 targets when an array of 4 crRNAs, Cpf1, and a GFP tag were transfected on a single plasmid. This is...~75% of neurons were co-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted...

... had a large genetic footprint (two cassettes + a booster cassette), their expression heterogeneity required...integratable cassettes, both with doxycycline inducible promoters (Fig. 2a). The first cassette contains ...the second cassette contains the P2A-linked accessory genes gvpNJKFGWV marked with Emerald GFP. These plasmids...control. We also added the toxin-antitoxin stability cassette Axe-Txe so that the plasmid could be maintained... that Anabaena mARGs did not require a booster cassette, either, allowing us to reduce the genetic footprint...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow...gating for the top quartile (or higher) of BFP and GFP producing cells. After FACS, return the cells to ...

...side of a stop cassette, upstream of an effector gene. In cells where the stop cassette is removed during...cells will express the effector or retain the stop cassette following recombination. Image from Isaacman-Beck...effector is expressed. In cells where the stop cassette remains, the effector is not expressed and the...attP sites to drive expression of either a stop cassette or an effector gene. HDVR (2X Hepatitis Delta ... Truncating the attP site upstream of the stop cassette results in decreased efficiency of recombination...preferentially leads to gene constructs where the stop cassette remains following recombination. This strategy...transcription activation systems, such as LexA/p65 or mCD8/GFP, to allow sparse labeling in a variety of fly cell...

...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...pSpCas9n(BB)-2A-GFP (PX461) - Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses...requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses...found to have a point mutation in the puromycin cassette that makes it less efficient in some cell lines...nick, rather than cleave DNA. Find pSpCas9n(BB)-2A-GFP (PX461). pSpCas9(BB)-2A-Puro (PX459) - This mammalian... It contains a point mutation in the puromycin cassette that makes it less efficient in some cell lines...

...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed.... https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article...reporter consisting of a quickly-degraded destabilized GFP variant fused to a 4X CDRE repeat. In addition to... Ca2+ (bottom panels) equipped with destabilized GFP calcineurin reporter (blue) and stained with PI (...death. Image from Diessl et al., 2020. Find the GFP reporter calcineurin activity here Diessl et al.,... They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison to intercalating...

...the targeted gene. Upon recombination, the GFP/NeoR cassette is inserted in place of exon 2. Thus, the ...recombinase. If GFP is under control of an endogenous promoter, you can use expression GFP to track cells...tag an endogenous protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab...In order to both knockout a given gene and insert GFP into the genome, we’d create a plasmid similar to...to the one shown below, where the sequence of GFP is cloned together with the Neomycin resistance (NeoR...gene is disrupted (knocked out) but the inserted GFP is expressed (knocked in). As seen in the above example...knockout/knock-in targeting vector designed to insert GFP into a given locus.   Conditional knockouts ...

...bacterial selection cassette for growth and cloning in bacteria and a mammalian selection cassette to screen for...cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). You can also upgrade...with an integrating vector expressing a selection cassette with 5′ and 3′ flanking sequences matching the...fusion proteins. Pick your promoter, selection cassette and visualization vehicle to conduct your custom...using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following the expression pattern... allow you to visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently...

...anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling...perform multiplex editing within a single expression cassette is advantageous to Cas9’s larger expression requirements...2: comparison of Cas 9 vs. Cas 12a expression cassettes when encoding multiple guides. While two guides...ability to target multiple genes within a single cassette provides increased simplicity for combinatorial...This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag fusions,...fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG...

...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning and...and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid...carries CRE recombinase and a puromycin resistance cassette. No time to produce virus? Get the ready-to-use...Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette. It can be used to produce a ~10-fold higher titer...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the...enabled scientists to engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity...Cas9 gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy...plasmid undergoes HR or SSA to reconstitute functional GFP, and the cells will turn green. Mashiko et al.,...

...including Standard PolIII-gRNA cassette, Csy4-cleavable cassette, and PTG cassette.    Find gRNA multiplexing...destination vector contains GFP, enabling you to select cells with high GFP expression. These cells have...smaller than constructs with multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors...aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid curing through "self-destruction" ...

...death. The team used this cassette to knock out genes, knock in a GFP cassette, and introduced site-directed...for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct repeats which can be ... selects for those cells that have removed the cassette through a second recombination event between the...

...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...introduced. Cre-mediated excision of the selective cassette minimizes the sequence added, leaving a 34 bp ..., however the development of the self-excising cassette (Dickinson et al., 2015) made this step as simple...homology arms to knock in large epitopes, such as GFP (Paix et al., 2014). A further development of this...efficiencies, obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence...Engineering with a Self-Excising Drug Selection Cassette. Genetics 200:1035–1049 . https://doi.org/10.1534...

...be another method to assess transfection success. GFP is often used as a reporter and we will be covering...up positive traits (i.e. the plasmid contains a cassette that will make cells resistant to a toxin), whereas... a negative trait (i.e. the plasmid contains a cassette that will make cells sensitive to a toxin). In...

...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...GTPase in division) GFP-Mff (outer membrane protein) Late Endosome mCherry-Rab7A GFP-Rab7A     ...result in a translational fusion of the antibiotic cassette to the gene of interest. The gene to be expressed... missing codons from the antibiotic resistance cassette, a ribosome binding site, SacI restriction site...heterologous promoter outside of the Multisite Gateway® cassette, you can specifically express your new chimeric...protein Knock-off, is made up of three components: a GFP protein that binds to the coding region of a reporter...

... in E3 and insertion of GFP expression cassette. Bunz 179202 pAd5-B6/7 ∆E3-GFP Adenoviral A block for ...regions present in pAd5-B6 ∆E3-GFP and pAd5-B7 . Bunz 179203 pAd5-B1-deltaE1-GFP Adenoviral A block for the...1 with deletion in E1 and insertion of GFP expression cassette. Bunz 16402 pShuttle Shuttle For production...production of GFP-trackable viruses containing transgene under CMV promoter Vogelstein 16407 pAdEasy 2-GFP beta-gal...with large inserts Vogelstein 179201 pAd5-B6 ∆E3-GFP Adenoviral A block for the AdenoBuilder genome assembly...Vogelstein 16404 pAdTrack Shuttle For production of GFP-trackable viruses containing transgene under a chosen...beta-gal Shuttle Test plasmid that contains β-gal and GFP; contains ∼10Kb genomic DNA stuffer Vogelstein 18703...

...experiment, consider co-transfecting with GFP. This allows you to sort for GFP-positive cells and to enrich for...for example, plasmid with a puromycin resistance cassette, such as PX459). After you co-transfect the CRISPR...