...assays to select both over- and under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable... Pages Browse Fluorescent Protein Plasmids Find Mammalian Expression Vectors ...and PCR, depending on the level of endogenous expression. We highly recommend the use of an electronic...ampules each of 4 clones with different levels of expression of our gene of interest. Advantages of the agarose...isolate individual cell clones with different expression characteristics from a population of transfected...Time CRISPR User) The Challenges of Cell Culture Plasmids 101: Mammalian Vectors Resources on Addgene.org...the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also...

...Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts ...Nathaniel Roquet from the Lu Lab at MIT developed plasmid based systems that report the order in which specific... action. AAV: A Versatile Viral Tool for Gene Expression in Mammals Adeno-associated viruses (AAVs) are... plasmid...

...chemistry! The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly...advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists...have two parts: (1) a genetically encoded “tag” expressed as a fusion with your favorite protein and (2)... Generate Fluorescent Protein Fusions Request Plasmids from the Michael Davidson Collection Find Fluorescent...photons. For example, moving from transient overexpression of protein fusions to gene-edited cells can...

...of the second plasmid can be done in one day in one assembly reaction. This second plasmid saves you a ...alerts when plasmids become available.  We’ve also had some requests for variations on the plasmids we’ve developed...and Myers for speaking with us! Find the Plasmids: Plasmids for protein tagging with CRISPR Additional... available at Addgene. For more details on the plasmids that are currently available along with detailed...several projects and this is one related to gene expression. One goal of the ENCODE project is to look at...system, what we are doing is adding in a second plasmid to target an epitope tag onto the transcription...second vector is a homology directed repair donor plasmid with homology arms and a selection marker. If things...

...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic... gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed them along with Cas9 in the T-cell line 2D10. ...of the LTRs joined together. Clonal Cas9/gRNA-expressing cell populations were also immune to HIV-1 reinfection...cycle progression, or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 .... For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, ...preventing re-infection would require sustained expression of Cas9/gRNAs, which could result in higher rates...the cells infected with HIV-1 only after stable expression of Cas9/gRNA. Viral production is higher in this...

...endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...production. Whether redesigning viral packaging plasmids, investigating different viral production strategies...

... as a donor plasmid containing the transposon, along with a helper plasmid that expresses the transposase...lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with plasmid-based...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...the dose of plasmid the cells receive over the whole reprogramming period and the plasmid will get diluted...larger plasmids have lower rates of transfection. Minicircles: Minicircles are like mini-plasmids. They...

...Browse fluorescent protein plasmids and resources See all Jaffrey lab plasmids ... cellular processes by transforming cells with plasmids encoding aptamer-tagged RNA and later introducing...aptamer downstream of a known promoter. Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment...aptamers able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...leading to conformational changes that control the expression of coding RNA. Spinach-based riboswitches can...SELEX, the RNA pool was cloned into bacterial expression vectors to create a library. Bacteria were transformed...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely...

...red light instead of blue to excite a molecule of GFP. Because red light has approximately half the energy...light, two photons of red light are needed to excite GFP compared to only one of blue. Only at the focal point...infinitely small point of light – think of a single GFP molecule – will appear as a fuzzy 200-300 nm spot...Resources Online Microscopy Primer on the Molecular Expressions Website Other major microscopy manufacturers ... Find Empty Fluorescent Protein Backbones Find Plasmids for Optogenetics ...

...Turquoise (CeNL; Plasmid #85199), Yellow (YeNL; Plasmid #85201), Orange (OeNL; plasmid #85202) and Red ... Researchers have been sharing plasmids ever since there were plasmids to share. Back when I was in the...specific strategies and tactics for getting the plasmids he wanted. Addgene makes it so much easier to ...kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk...reporter was used to sort successfully infected/expressing cells by flow cytometry. The isolated cells were...fluorescent protein that could be co-imaged with GFP using the same wavelength for excitation. The result...CyOFP1-NLuc-CyOFP1; Antares). Direct injection of plasmids for Antares or firefly luciferase into mouse livers...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that... you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over...over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the... that restrict expression to this cell type. To this end, they constructed AAV plasmids bearing one of...bacterial and mammalian mCyRFP1 or CyRFP1 expression plasmids, FRET constructs, and CaMKII and RhoA sensors...

...the IP in parallel with both a sample known to express the protein of interest, such as a sample transiently...include knockout cell lines or tissues that do not express the protein. With this control set, you should ...In cases such as this, an epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest ...interactions of interest. Tagged proteins are typically expressed at much higher levels than endogenous proteins...antibody posts  Resources on Addgene.org: Find plasmids encoding recombinant antibodies at Addgene ...

...summer or just a bright new plasmid. One area with lots of bright new plasmids is our Fluorescent Biosensors...are generated by inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled...Figure 2: iGluSnFR3 displays enhanced brightness, expression, and sensitivity compared with earlier SF-iGluSnFR...SF-iGluSnFR. Image from Aggarwal 2022.   Find iGluSnFR3 plasmids here! Find viral prep here: pAAV.hSyn.FLEX.iGluSnFR3.... 2022, under CC-BY-NC license.   Find JEDI-2P plasmids here! Find viral preps here: pAAV-EF1a-DIO-JEDI.... By fusing ITAM to an RTK of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence...et al. 2022, under CC-BY license.   Find pYtags plasmids here!   Farahani PE, et al. pYtags enable spatiotemporal...

...markers were not increased compared to a control AAV-GFP. Not only did the AAV-SaCas9-sgRNA constructs mediate...capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation...activation. Split inteins have also been used to express base editors. References Chew WL, Tabebordbar ...HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep 5: . https://doi.org/10.1038...bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038...Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ...

...with mixed expression, with the expression measurable through an epitope tag such as FLAG/His/GFP. Fluorescent...epitopes. Detection of overexpressed (tagged or untagged) protein When expression plasmids are available, or...flow cytometry, and mRNA expression.   If your protein of interest is expressed at similar levels in ... involves the transient transfection of expression plasmids in a given cell line. For flow cytometry, ...differences between endogenous expression and transfected overexpression, this approach cannot confirm ...caused by overexpression. Additionally, if the cell line used has endogenous levels of expression, this can...preferred. The overexpression approach is therefore best suited to cell lines without any expression of the target...

...rewards could be substantial: Plasmid tRNA Synthetase Expression Host pMAH-POLY Polyspecific aminoacyl-tRNA...Resources on Addgene.org Find Plasmids for FRET Browse Empty Plasmids to Fluorescent Protein Fusions ...with natural chain termination at stop codons, expression levels of these tagged proteins are often very...oocytes, mainly). Nevertheless, tRNA suppression plasmids are available from depositing labs that have mastered...Fig. 1) which can be carried out using Addgene plasmids. His-tag labeling reagents You may already have...protein, permeabilized cells  Cy5NTAc 32 25-45 GFP (fluorescein is similar)   Lumio Red Tc: CCPGCC 48...

...Dangers of Using GFP for Protein Localization Resources on Addgene.org Find Plasmids for Mammalian Cells...along with Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal...

...transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase ...different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles... and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into ... retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific safety... for a researcher to come into contact with than GFP. Similarly, if the viral vector carries an shRNA ...

...reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP target plasmid and pX330-sgRNA plasmid (Mashiko et...pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids for gRNA...site (MCS) between EGFP fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA...pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T... mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA." Scientific reports...fluorescent protein (EGFP) reconstitution. (a) Scheme of validation for DSB mediated EGFP expression cassette reconstitution...halves of EGFP to recombine by homology directed repair, and resulting in the expression of EGFP. Using ...

...using a plasmid with multiple, individual expression cassettes, having the genes expressed from the same...genes. Plasmid Name Multicistronic Element Expression Type MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP... for each gene that is expressed, multicistronic vectors simultaneously express two or more separate proteins...cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or...multicistronic plasmids ensure that any cells that are positive for your marker should also be expressing your ...want to express multiple genes in the same cell. Although it is possible to drive co-expression by using... a portion of the plasmid is packaged for viral delivery, or the relative expression levels between two...

...methylation in mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced... are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation...modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear....that help package DNA, were associated with gene expression or silencing. To alter DNA modifications, researchers...Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and...well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks...cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation...

... Nuc-flox(mCherry)-EGFP 50457 AAV2 pAAV-hSyn-DIO-EGFP Recombinases Plasmid Serotype Name 87306...the entire collection! Plasmid Serotype Name 51502 AAV5 pCAG-FLEX-EGFP-WPRE 114471 AAV1, AAV5 pAAV-Ef1a-fDIO...constructs from Christopher Harvey’s lab. These plasmids localize opsins to the soma to avoid off target...neighboring neurons. We have also packaged hChR2(H134R) plasmids from Karl Deisseroth’s lab, which includes pAAV-hSyn...H134R)-EYFP that is both Cre and Flp dependent. Plasmid Serotype Name 124603 AAV9 pAAV-EF1a-DIO-ChrimsonR-mRuby2...only interpret their results, but also confirm expression in target cells. While the various AAV serotypes... can be used to achieve different patterns of expression, researchers often need to determine these transduction...

...factor. Bulkier tags, such as GFP, may interfere with protein expression or function in live cells. Fluorophore...cell and protein analysis products. You can find plasmids deposited by Promega here.    References and ...More resources from Addgene Fluorescent protein plasmids and resourcesA practical approach to choosing ...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...about a recent plasmid deposit please sign up here.   Listen this episode of Hot Plasmids! Light controls...available in a mammalian expression vector (pcDNA3) and a bacterial expression vector (pRSETB). Fabritius...) in real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry...Hahn's lab at the University of North Carolina (plasmids at Addgene). It consists of two domains, LOV2 ... the same number of fluorophores. You can find plasmids containing the cobalamin binding riboswitches ...

...resources on addgene.org Find Optogenetics Plasmids Find Biosensor Plasmids Visit the Fluorescent Protein Guide...very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight but detectable change...Frank Hayward et al., 2024). They deposited their plasmid with Addgene, hoping to inspire others to explore...one-component magnetoresponsive system that could be expressed and purified. Although a ~1% change in signal ...” magnetoresponse was clearly visible whether expressed in E. coli, in mammalian cells, or in purified...   Video 1: Fluorescence movie of E. coli expressing MagLOV while a magnet is waved under the sample...Lab is eager to hear from you. Find the MagLOV plasmid here!   References and resources References Duret...

...receive transfer plasmid DNA that you can use along with lentiviral packaging plasmids to create your own...rough approximations of the total number of CRISPR plasmids for that particular organism available at Addgene...helped distribute many of these technologies in plasmid form and are proud to have facilitated their fast...fast adoption. However, in many cases the plasmids themselves are only the starting point for the production...infections with all of our viral preps and host our expression data (western blots or fluorescent microscopy...nickase for genome editing. You can also enhance expression of your gene of interest with a variety of Cas9... use; after receiving a pooled gRNA library in plasmid format, a researcher must amplify the library, ...

...labs’ plasmids, including protocols, check out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes...looking for a broader range of CRISPRs plasmid tools, find more plasmids, CRISPR technology guides, FAQs, and...Within target cells, Cas9 machinery is transiently expressed and binds to target DNA. In the final step of ...large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral particle. The earliest...description of their experimental protocol. The three plasmids which have been deposited to Addgene are: pAdSh.PGK.Cas9...pAdSh.PGK.Cas9, pAdSh.U6.gRNAS1, and pAdSh.U6.gRNAGFP. Gonçalves says that advantages of AdVs include their...guide RNA constructs, pAdSh.U6.gRNAS1 and pAdSh.U6.gRNAGFP, and also Adeno Cas9 and Adeno EA. Or if you're...

...et al, 2012, Bindels et al, 2017). The plasmid for co-expressing mTurquoise2 with mVenus as a reference...mammalian cells is transiently transfected with a plasmid containing a fluorescent protein (fusion) there...dish. This is caused by the stochastic nature of plasmid uptake by cells. To correct for this variation,...variation, a reference fluorescent protein that is expressed at an equal level (Goedhart et al, 2011) can be used... The practical brightness assay (employing co-expressing fluorescent proteins or using endogenous tagged...Find Empty Backbones with Fluorescent Tags Find Plasmids for Subcellular Localization Browse the entire... QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number, the higher the theoretical...

...Unpublished or pre-publication plasmids": Unpublished plasmids or plasmids that will not be published are...and entering a plasmid name,plasmid type, and plasmid description/experimental purpose (right). On the...with your plasmids, and adding all the plasmids at once increases the likelihood that the plasmids are given...easy for you to share plasmids. To achieve this goal, we will archive any plasmids you've deposited with...Spreadsheet". Start your plasmid deposit There are two options to begin your plasmid deposit. Figure 2: Choose... Choose to submit plasmid(s) online or by using a spreadsheet. The plasmid information can be entered ...than 10 plasmids but we recommend using the spreadsheet option for deposits of 10 or more plasmids.   ...

... with plasmids expressing the GPCR of interest, a Gα-Rluc8 plasmid, and the appropriate Gγ-GFP2 and Gβ...effects.  Bryan Roth’s lab created a collection of plasmids called TRUPATH to interrogate 14 of the 16 non-visual...TRUPATH be used for? The Roth lab used the TRUPATH plasmids in a number of different experimental approaches...drugs to further understand their mechanism. These plasmids are available as a collection, facilitating screening...Addgene.org Browse the catalog of fluorescent protein plasmids Find biosensors for your experiments ... acceptor GFP2-Gγ chimeras, and two untagged Gβ constructs. For the Gγ constructs, the GFP2 tag is fused... Gα-Rluc8 and Gγ-GFP2 will be in close association, resulting in a high level of GFP2 emission. Following...