...Addgene. I am currently working on the QC/QA team as a DNA Sequencing Intern. The application process When ... the submitted sequences were a concern to the plasmid function. The steps of the training process gave...very approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition to my responsibilities...

...want delivered to your target cells. This delivered DNA/RNA can then be either permanently integrated into...sequence for degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing temporarily...transfection and random genomic integration of a plasmid. Viral transduction is also generally less toxic...the virus are now delivered in other forms (mRNA, DNA, etc.) to raise immunity. Viral vectors are being...

...macromolecules used in scientific research such as DNA or protein. It comes as no surprise then that stability...Base modifications What is more stable than RNA? DNA! One way to make your gRNA more efficient on a budget...ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein...ready-to-use viral preps of select CRISPR lentiviral plasmids Additional resources on the Addgene Blog  How...

...with some plasmids. You just need three plasmids to start making AAV:  the packaging plasmid which contains... transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases each and AAV plasmids have two ...of an AAV transfer plasmid. Parts shown in green are key elements of an AAV plasmid. Parts shown in grey... elements of an AAV plasmid First let’s talk about the key parts of an AAV plasmid. Inverted terminal ...transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid harbors non-coding...Transfer Plasmid video!   Now you’ve got a basic understanding of some common AAV plasmid elements!...structural and packaging genes, the adenoviral helper plasmid which contains the proteins needed for the virus...

...Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria." Plasmid 51.3 (2004): 246-255. PubMed...exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate off-target mutations...a mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile... These two steps are (Figure 1): The initial plasmid integration step (the so-called “single crossover...versatile, synthetic MCS (designed with the help of the DNA tuner program) (Latynski and Valentovich 2014). We... an allelic exchange experiment? Find the pTOX plasmids here! Continued development of new allelic exchange...PMCID: PMC4513841. Latynski US, Valentovich LN. "DNA tuner: a computer program for the construction of...

...giving you more choice in how you receive plasmids — as DNA or bacterial stabs — ‌and reinventing our ...accelerating scientific discovery. Over 150,000 plasmids empowering researchers worldwide. Countless breakthroughs...countries enriched our repository with 15,020 new plasmids — a testament to the scientific community's commitment...next-generation sequencing, setting new standards for plasmid quality control. Through our partnership with the... viral vector options. Almost any AAV transfer plasmid in our repository can now be packaged on demand...sequences and characterization data for over 150,000 plasmids, empowering bioinformaticians, data scientists...developers to unlock the potential of our extensive plasmid database — because we believe that innovation flourishes...

...components: GBPa-VP16 (activation domain); GBPb-GAL4 (DNA-binding domain); and a UAS-driven luciferase reporter...systems. T-DDOGs can also be designed with other DNA-binding domains, including the commonly used LexA...23953120. PubMed Central PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan...2015): 1334-1341. PubMed PMID: 26258682. Find plasmids from this publication at Addgene. 3. Kirchhofer...

... function (Pan et al., 2018). cDNA libraries, which consist of plasmids that over-express coding sequences...been most widely used to create specific edits in DNA, there has also been significant work on constructing... any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a transcriptional activation ... activators, similar tools were made using other DNA binding proteins such as zinc fingers (ZF) and TAL... several popular CRISPR-based tools for altering DNA sequence or regulating gene expression.     References...they want to activate, we would recommend using a cDNA overexpression vector rather than going through ...and deliver when compared to gRNAs. Additionaly, cDNAs cannot be used to study in cis regulation and also...

...introducing double stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic...homology-directed repair (HDR), a distinct cellular DNA repair pathway. Accomplishing this usually involves...involves simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide...at all, such as when you are editing a non-coding DNA region? To this end we developed a technology called...Ribonucleoproteins Resources on Addgene.org Browse All CRISPR Plasmids Check Out Our CRISPR Guide Pages Find Validated...

...incorporate new information into DNA. This also requires an exogenous DNA template. HDR, however, is a very...so-called base editor Cas9, which makes DNA changes in the absence of dsDNA breaks (Rees et al., 2018). For C...made it easier than ever both to engineer specific DNA edits and to perform functional screens to identify...efficacious to target the coding or non-coding strand of DNA. As a rule of thumb, we avoid target sites that code...CRISPR Basics with Our CRISPR Guide Find CRISPR Plasmids for Your Research Find CRISPR Pooled Libraries...technology is usually accomplished by Cas9-mediated dsDNA breaks: following a cut, the error-prone nature ...although this is rarely done). When targeting a dsDNA break for HDR, the choice of target site is far ...

...techniques are still used and efficiently transfer plasmid DNA efficiently into dividing immortalized cells ...virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing all the structural genes...crossover event between the vector plasmid and the packaging plasmid caused the Psi sequence to be placed...expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone your gene of...usually 293T cells, with your transfer plasmid and your packaging plasmids. For this, you can use the transfection...reach their limits when it comes to transferring DNA into primary cells or non-dividing cells. To target... (Gag, Pol, Tat, Rev and Env) and the transfer plasmid encoding the insert of interest as well as sequences...

...flanking the DNA-binding cleft of Cas9, the fluorescent domains dimerize and prevent DNA binding in the... cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by the guide RNA (gRNA), while...unveiled a photoactivatable system to cleave a target DNA sequence (Nihongaki, et al., 2015) resulting in a... Additionally, high intensity exposure can cause DNA damage. Far-red light, however, can penetrate 5 mm...fluorescent reporter gene in mice using Minicircle DNA vectors encoding the FAST constructs. This proof-...stimulation, the pdDronpa moieties move apart, allowing DNA binding and cleavage. This technique was applied ...deactivated the protein’s ability to cleave targeted DNA, settling on photocaging the K866 lysine, as seen...

...will be able to request an AAV composed of an AAV plasmid in the Addgene repository and their choice of five... more.  What will be available?  Over 3000 AAV plasmids in the Addgene repository will be available for...Packaged on Request service. A small subset of AAV plasmids will have technical or legal restrictions that...both our depositors and requestors.  Requesting Plasmids available for Packaged on Request consideration...sequencing to confirm the identity of the packaged DNA and serotype. Please note that Addgene cannot perform...recommend you check the original paper of your AAV plasmid of choice for more information on its performance...performance.   Titers We expect titers for plasmids packaged in AAV1, AAV5, AAV8, and AAV Retrograde to be 1x1013...

.... pBLADEONLY: This plasmid contains only BLADE and is based on the pTRC99a plasmid. It does not include...AraC and other promoter systems, head over to our Plasmids 101: Inducible Promoters article.) Modifying AraC...team tested different linkers between VVD and the DNA-binding domain of AraC using a mCherry reporter downstream...reporter downstream of the PBAD promoter. To use this plasmid with your gene of interest, you would replace mCherry...PBAD promoter. If you already have a pBAD33-based plasmid expressing your gene of interest, all you have ... and shared with Addgene. You can find all the plasmids deposited from Di Ventura's lab, including the...nuclear export system (LEXY) here. Optogenetic plasmids from the Khammash lab can also be found here. ...

...Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to researchers in developing...Addgene-Seeding Labs Plasmid Grant Check Out Previous Addgene-Seeding Labs Plasmid Grant Awardees Listen...scientists who were awarded the Addgene-Seeding Labs Plasmid grant this year. These scientists work on projects...technology to engineer plants that are able to destroy DNA viruses, similar to the anti-phage CRISPR effect ...as the pET GFP LIC cloning vector and the pOKD plasmids. The recombinant Cry proteins will then be screened...HyPer3 (2) which he will co-express together with a plasmid encoding glycine oxidase. The goal is to determine...excited to see how all of the Addgene-Seeding Labs Plasmid Grant awardees' projects progress. Subscribe to...

...Browse fluorescent protein plasmids and resources See all Jaffrey lab plasmids ...winter day, you should be happy to learn that many DNA-based reagents are named for soup ingredients! From... 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...has been used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al.,...

... that a small DNA binding motif in MECP2 interacts with the minor groove of AT-rich DNA sequences to regulate...their MECP2-related plasmid tools with Addgene and share additional resources. Plasmid tools to facilitate...to partner with them to launch a Rett Syndrome Plasmid Collection and Resource Center for Rett Syndrome... Rett Syndrome in novel ways.  Addgene’s MECP2 Plasmid Collection and Resource Center will continue to...

...., 2019). AdVs are non-enveloped double-stranded DNA viruses, with a linear genome between 26–45 kb in...initiating the whole process – such as the viral DNA polymerase – it makes sense that vectorization strategies...strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted.... These genes’ functions are rescued by helper plasmids used during production and/or by genes present...made available by this deletion from the genomic plasmid can be used to house a recombinant sequence (transgene...late genes are usually present in the transfer plasmid for the majority of adenoviral vector systems used...

...s incredible.” Looking for CRISPR Plasmids? CRISPR-Cas9 Plasmids available at Addgene CRISPR-Cas9 References...Forbes respectively: "A Powerful New Way to Edit DNA" and "This Protein Could Change Biotech Forever."...Last month, the Lawrence Berkeley Lab's Jennifer Doudna and Eva Nogales presented the first detailed picture...

...multiple gRNAs on the same plasmid, you’ll make sure that each cell that gets the plasmid contains all of the...rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction...different purposes.   Gersbach Lab multiplexing plasmids: This plasmid set allows you to express 2-4 gRNAs, with...assembled to produce a plasmid encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 ...Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through...Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids can be used to express up to 8 gRNAs after...Yang Lab Single Transcript Multiplexing Plasmids: These plasmids are similar to the Csy4 polycistronic ...

...the edited strand and wild-type strand of the host DNA. When these strands hybridize, the nuclease can detect...account other elements, including those relating to DNA structure (e.g. epigenetic modification, bulges) ... et al. "High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity...Frock, Richard L., et al. "Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases... Crosetto, Nicola, et al. "Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing...Addgene.org View Our CRISPR Guide Pages Find CRISPR Plasmids for Your Experiments Get Ready-to-Use CRISPR Lentivirus...

...SnapGene powered plasmid maps at Addgene  Three factors make our SnapGene powered plasmid maps far easier...easier for you to share plasmid information with colleagues, or explain plasmid features to a lab member...sequences, & downloadable .gb & .dna files Once you’ve identified a plasmid that you want to learn more about... on the main plasmid page under the "Resource Information" section. Analyze your plasmid sequence For ...over from our users: "Please, please improve your plasmid maps!" After thoughtful design, vetting, and tweaking...tweaking, we’re excited to announce that our plasmid and sequence displays are now powered by GSL Biotech's...software and extensive feature library, our updated plasmid and sequence displays are now much easier to interpret...

...articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions...genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment...with expression, though it is possible for the plasmid to still express without the G. Because the G is...additional 'G'. Q3: What is the maximum amount of DNA that can be inserted into the genome using CRISPR...marker to select transfected cells (for example, plasmid with a puromycin resistance cassette, such as PX459...related to the most frequently requested CRISPR plasmids at Addgene Find protocols and gRNA design tools...by a variety of labs and optimized for specific plasmids Links to different software to help you identify...

... of CRISPR components into target cells, genomic DNA from both edited and unedited (control) populations...from single or multiplex CRISPR-Cas9 double-strand DNA breaks using SpCas9. More features will be added ...the user-defined 17-23 nucleotide sequence of the DNA-targeting region of the guide RNA, excluding the ...Addgene.org Check out our CRISPR guide Find CRISPR plasmids for your research Find other CRISPR software resources...

...independently searched for this unique sequence of DNA and both identified lymphocyte antigen 6 complex ...receptor for PHP.B by looking for a unique sequence of DNA that was present in mice whose CNS was transduced...chromosome. Of the genes located in this stretch of DNA, Ly6a seemed the most likely candidate due to its...permissive and non-permissive mouse strains. PHP.B plasmids and PHP.eB viral preps are available from Addgene...

...Resources on the Addgene Blog: Plasmids 101: The Promoter Region Plasmids 101: Cre-lox Light-Switchable...protein, the repressor protein is removed from the DNA. With the repressor protein absent, transcription...Promoter Subtype Promoter Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA promoter AlcR pGGA008...use light to regulate transcription. Red flame plasmid pDawn contains the blue-light sensing protein YFI...Systems for E. coli Browse other blog posts from our Plasmids 101 series Resources on Addgene.org: Inducible...Expression Systems at Addgene Cre-lox Guide and Plasmids     ...

...and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G. Find...wide range of applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB... top requested plasmid from 2016. You may notice the “V2.0” at the end of this plasmid which indicates...prior version of this plasmid. Indeed the first version is the 15th most popular plasmid of all time, but ...envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini....1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...Cas9, this plasmid expresses Cas9n (a D10A nickase mutant) to nick, rather than cleave DNA. Find pSpCas9n...

...analyzing the plasmid and viral DNA sequences and expression profile at multiple steps from plasmid amplification...to flip back into the inverse orientation (See Plasmids 101: FLEx Vectors). While this strategy greatly...recombination that are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means that...of this finding, we now include guidance on the plasmid page of DIO/FLEx AAV vectors where this occurs ... mechanism of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of ...Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts. Human Gene Therapy 31:664–678. https://....2020.618020 Additional resources on the Addgene blog Plasmids 101: FLEx Vectors Important Considerations When...

...bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038/nature09523...capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation...Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156:935–949. https://doi.org/10.1016/j.cell...Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ..., Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA (2019) CasX enzymes comprise a distinct family...

...this case, plasmid copy number is the input and gene expression is the output. The input, plasmid copy number...to decouple gene expression and plasmid number. In a suite of plasmids modified from pSC101 with copy ...of all types of plasmids, the stabilized promoters were also tested in different plasmid backbones that...stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, ...RepA is a plasmid replication initiator protein, they could alter copy number of the plasmid by altering...genome or a plasmid. Thus, a stabilized promoter can allow a system to be moved from a plasmid to the genome...special collections page Download the Plasmid 101 eBook See all plasmids deposited by the Voigt lab ...

...Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent Protein Guide...associated publication for the specifics of your chosen plasmid. Types of biosensors When choosing a biosensor...biosensors to follow processes such as protein or DNA binding, enzyme or transcriptional activation, conformational...

...γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (Vesicular...in trans by specific plasmids. This method, therefore, reduces the number of plasmids that are required ...encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the packaging cell...do many γ-retroviral plasmids have similar names? The nomenclature of the plasmid backbone is meant to...components are needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest..., which facilitate integration of the transfer plasmid sequences into the host genome. Typically, it is... genome upon viral transduction. Most transfer plasmids available from Addgene were derived from MoMLV...

...urgent requests for access to coronavirus-related plasmids and to receive important coronavirus-related deposits...shipping staff agreed to come in and start getting plasmid deposits into the collection and to start shipping...support Addgene now, when their distribution of plasmids for COVID-19 research is accelerating essential...’ve recently piloted Addgene’s new cloning grade DNA service, and viral vector distribution is recovering...-related slowdown. More scientists are sharing plasmids and other reagents every day, and this crisis ...

...the same page in terms of the vocabulary--DNA, protein, plasmid, gene, etc. Though I talked about CRISPR...kid’s face when you’ve shown them how to isolate DNA from a strawberry, or demonstrated that they’re living...out our comprehensive CRISPR guide Find CRISPR plasmids and resources ...

...conjugative mobilization of broad host-range IncQ plasmids." Plasmid 62.2 (2009): 57-70. PubMed PMID: 19465049...page Deposit microbiology plasmids with Addgene Find synthetic biology plasmids ...family-RP4 conjugation system. This system can deliver plasmids to both Gram-positive and Gram-negative cells.... Wang lab further developed a modular suite of plasmids (pGT) with various replicative origins that can...genera across four phyla that have taken up the plasmid through conjugation in vivo studies. However, they... tools for microbiome studies.  Find the MAGIC plasmids here Paper-based synthetic biology approach to...difficile. As mRNA expression cannot be identified with DNA-based qPCR methods, the ability to detect mRNA transcripts...

...Addgene blog Plasmids 101: Origin of Replication Plasmids 101: What is a Plasmid?  Plasmids 101: Transformation...host range in plasmids? Not all plasmids were created equally. Only a handful of plasmids have been identified...range plasmid with only one ori and a broad host range plasmid with multiple oris that vary in DnaA-box ...broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001  Meyer...define plasmid host range, identify a few of the features that confer broad host range in plasmids in nature...recombinant vectors.  A plasmid’s host range is determined by the different hosts that a plasmid can transfer to...influence the plasmid’s ability to establish and maintain itself within host cells.  The plasmid has multiple...

...This construct will serve as the repair template plasmid to create fluorescent and 3xFLAG-tag fusion proteins...proteins. The repair template and Cas9-sgRNA plasmid is then injected into the germline of young adult ....115.178335. [Epub ahead of print] PubMed. Find plasmids from this publication at Addgene. Other strategies...10.1038/nmeth.2641. Epub 2013 Sep 1. PubMed. Find plasmids from this publication at Addgene. Scalable and... and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans...genetics.114.170423. Epub 2014 Sep 23. PubMed. Find plasmids from this publication at Addgene. Additional...

...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...easily assemble multiple DNA fragments. This system has been used to construct plasmids for use in model organisms...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        AAV delivery... Figure 1: The base editor is split into two plasmids and co-transfected into cells for reconstitution...overcome this overlap, Yiyang Gong’s lab created two plasmids containing a red-shifted Ace-mScarlet, a fusion...channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019. PubMed...

...Recovering Plasmid DNA from Bacterial Culture Recovering Plasmid DNA from Bacterial Culture You may also...concentrated plasmid DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial...high concentration of clean plasmid DNA. To improve the purity of plasmid DNA purified without a kit it ...isopropanol to precipitate the plasmid DNA. Either spin to pellet the DNA or apply the solution to a column...How to purify plasmid DNA from a bacterial culture, including protocols, tips, and FAQ Protocols... new window) Promega sell kits for isolating plasmid DNA in quantities as low as a few micrograms to as...describe the general procedure for purifying plasmid DNA from bacterial cultures. If you will be using...

...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... use; after receiving a pooled gRNA library in plasmid format, a researcher must amplify the library, ...