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We narrowed to 255 results for: tre promoter

Showing: 1 - 50 of 255 results

  1. Tetracycline Inducible Expression

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    Collection
    ...unable to bind the TRE. In the presence of tetracycline, rtTA binds the TRE, promoting gene expression. ...and EGFP. Pbi contains a TRE between two minimal CMV promoters. None Pbi (TRE, miniCMV) Bert Vogelstein...Response Element (TRE) typically features seven copies of tet O upstream of a minimal CMV promoter, and other...introduced the TRE, placing seven copies of tet O upstream of the minimal CMV promoter. In the absence...may also like... Blog: Inducible Promoters Blog: Repressible Promoters Mammalian shRNA Plasmids Collection... of tetracycline, tTA binds to the TRE and its VP16 domain promotes gene expression. In the presence of...fold induction of the gene of interest. Tight TRE promoter or P Tight : reduced background expression and...

  2. Plasmids 101: Repressible Promoters

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    Blog Post
    ... are repressible promoters regulated? Like inducible promoters, repressible promoters can be regulated...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...natural and engineered repressible promoters. Chemically repressible promoters Tet-Off In its native context... function well in mammalian cells, and TRE-containing promoters can be used in the repressible manner ... pADH1 displays low promoter activity (~20% activity of the strong yeast promoter pTEF). As ethanol accumulates...that promoter is active. In this way, you can interrogate the activity of uncharacterized promoters. Similarly... QUAS promoter, and the QF transcription factor cannot drive transcription from the UAS promoter. This...

  3. Plasmids 101: Inducible Promoters

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    Blog Post
    ... on repressible promoters. How are inducible promoters regulated? Inducible promoters can be regulated...steroid regulated promoters commonly used in plant research. Promoter Subtype Promoter Example Activator...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...will be produced. Many common promoters. like CMV, EF1A, and SV40 promoters, are always active and thus ...of a promoter. Below, you’ll find a short description of these three types of inducible promoters, and...Many of these promoter systems are available at Addgene! Chemically inducible promoters Chemically regulated...regulated promoters are among the most common inducible promoters. The positive inducible tetracycline ...

  4. Plasmids 101: The Promoter Region – Let's Go!

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    Blog Post
    ... trying to pick your perfect promoter! Eukaryotic Promoters   Promoter   Primarily used for RNA     .... In practice, the term "promoter" describes the combination of the promoter (RNA polymerase binding site...recognize and bind to specific promoter elements. This means that the promoter present in your plasmid backbone...RNAP II promoter, whereas small RNAs (such as shRNA) are transcribed from the RNAP III promoters. This ...post on viral vector parts. Promoter specificity Aside from choosing a promoter based on type of RNA transcript...cell types or organisms, promoters must be similarly variable. Bacterial promoters only work in prokaryotic...require unique promoters and there is very little crossover. Generally speaking, promoters in bacteria are...

  5. Quick Guide to All Things Lentivirus

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    Blog Post
    ...system a Tet-response element (TRE) is placed upstream of the promoter in your transfer vector. In the...and the promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been...the control of two different promoters. Both the single and dual promoter transfer vectors can be used...containing the rev response element (RRE), an internal promoter, and the desired gene (transfer vector plasmid...entry site (IRES) and expressed under the same promoter. Alternatively, your gene and the marker can be...cell cycle, a transfer vector with an inducible promoter (see below) should be used. Inducible lentiviral... expressed by your transfer vector) binds to the TRE and activates the expression of your gene. The addition...

  6. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

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    Blog Post
    ...with which promoters, what part of a gene enhancers like to interact with, and how many promoters or genes...published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation, but enhancers...its capture rate of chromatin interactions at a promoter in the well-characterized beta globin locus. The...a cell line model, the Xu Lab tracked enhancer-promoter interactions during erythroid differentiation.... tre...into pieces small enough to be pulled down via streptavidin IP. Biotinylated dCas9 goes along for the ride...

  7. Synthetic promoter AAVs for cell-type specific expression in retinal cells

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    Blog Post
    ...finding retinal cell-type specific promoters. Most existing promoters express in a broad number of cell... occurring promoters to drive transgene expression, the Roska lab built synthetic promoters from scratch... for designing synthetic promoters  To build a library of synthetic promoters, the Roska lab used four... containing the active promoters at Addgene. Performance of synthetic promoter AAVs in mice All of these...class of cells  Of the four different promoter designs, promoters from strategy #4 had the highest rate... of synthetic promoters replicated the expression specificity of the wild-type promoters they were derived...natural promoter that was active in the outer part of the retina.  Find the AAV synthetic promoter plasmids...

  8. Luciferase Plasmid Collection

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    Collection
    ...of 5' promoter/enhancer regions Joshua Mendell 60323 pGL4.23-GW Firefly Insertion of 5' promoter/enhancer... reporter vector. Insertion of 5' promoter/enhancer regions upstream of Firefly luciferase. Renilla luciferase...tool for scientists. Regulatory elements such as promoters, enhancers and untranslated regions, or shRNA ...Jorge Ferrer 16539 pBV-Luc Firefly Insertion of 5' promoter/enhancer regions Bert Vogelstein 12178 pIS0 Firefly... Renilla luciferase under the control of a CMV promoter is present for normalization William Kaelin 71248...71248 pXPG Firefly Insertion of 5' promoter/enhancer regions. Mutations within the luciferase gene allow...

  9. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

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    Blog Post
    ...stabilized promoters. In contrast to unregulated promoters that are always on, stabilized promoters incorporate...constitutive promoter upstream of GFP increased expression of GFP six-fold, but a stabilized promoter increased...with the stabilized promoter while gene expression from the constitutive promoter did not. Similar levels...Segall-Shapiro et al., 2018). Designing stabilized promoters To keep gene expression stable regardless of copy...if you introduce negative regulation by using a promoter that is responsive to a repressor protein, the...level constant. In the case of the stabilized promoters from Segall-Shapiro et al., they chose a TALE ... addition, TALEs bind non-cooperatively to the promoter region, which is predicted to be required to decouple...

  10. Viral Vectors 101: Parts of the AAV Transfer Plasmid

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    Blog Post
    ...interest. Promoter The promoter drives the expression of the AAV transgene. On a plasmid map, promoters are ...location. Promoters can also drive different levels of expression. For example, CAG is a strong promoter that...synapsin 1 promoter is neuronal specific (Haery et al., 2019). Most AAV transfer plasmids have a promoter, but...are upstream or 5’ of the gene they control. Promoters can be organism or tissue type-specific, which can...nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...for the perfect AAV tool Learn about synthetic promoter AAVs for cell-type expression in retinal cells...the end of the AAV genome, just upstream of the final ITR and downstream of the WPRE. TL;DR? Watch our ...

  11. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

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    Blog Post
    ...contains BLADE, the PBAD promoter, and a mCherry reporter downstream of the PBAD promoter. To use this plasmid...on AraC and other promoter systems, head over to our Plasmids 101: Inducible Promoters article.) Modifying...domain of AraC using a mCherry reporter downstream of a PBAD promoter. While none of the constructs gave as...protein that controls transcription from the PBAD promoter. “AraC is widely used in synthetic biology, biotechnology... pTRC99a plasmid. It does not include the PBAD promoter. If you already have a pBAD33-based plasmid expressing...previously cloned gene of interest, under the pBAD promoter, can be controlled with light without the need...superfolder GFP instead of mCherry under the PBAD promoter. They created a bacterial lawn on an agar plate...

  12. Hot Plasmids - November 2023

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    Blog Post
    ... expressed under promoters like the tet-responsive element (TRE) or the cFos promoter. For example, neurons...inducibly express tagged iPAK4 from different promoters, specific time points appear as fluorescent bands...new way to record and quantify the dynamics of promoter activity in single cells. Find iPAK4 plasmids ...targeting components are interchangeable. Image from Strebinger et al., 2023, reused under CC-BY 4.0 License....medical applications.  Find DIRECTED plasmids here! Strebinger, D., et al. (2023). Cell type-specific delivery...moving to and from the nucleus, ER, mitochondria, stress granules, and more. For added precision, the authors...neurons treated with a phorbol ester activator gave fluorescent signal in the ticker tape from cFos::GFP-iPAK4...

  13. Viral Vectors 101: AAV Variables That Matter

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    Blog Post
    ...your experiment site or circuit can occur. Promoters The promoter controls how the gene of interest will ...selective, or inducible.  Ubiquitous promoters  These often-strong promoters offer the most flexibility for ...vitro) or months (in vivo).  Selective promoters Selective promoters, such as synapsin, glial fibrillary ...International License.   Inducible promoters  This type of promoter allows you to decide when to turn ...three key variables of an AAV vector: serotype, promoter, and titer.     Figure 1: Flow chart depicting...depicting a) steps for selecting serotypes and promoters to test in your AAV system and b) steps for testing...testing and optimizing your serotypes, titer, and promoter. Note that several steps in testing are effected...

  14. Plasmids 101: Protein Expression

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    Blog Post
    ...of constitutive and inducible promoters. Inducible promoters are extremely useful if a desired protein ... of the galactose inducible promoter (GAL). Other commonly used promoters include the phosphate and copper... Blog Learn about inducible promoters Learn about repressible promoters Check out protein tags Resources...designed for mammalian expression utilize viral promoters (SV40, CMV, and RSV) for robust expression post-transfection... of these vectors is controlled by a different promoter resulting in different levels of expression from... vectors, pET, under the control of the T7 lac promoter and induced by lactose, provides the highest level...copper inducible PHO5 and CUP1 promoters respectively. Yeast cells are grown in well-defined media and...

  15. Plasmids 101: The protein expression toolbox

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    Blog Post
    ...more elements of control. Tet promoters Tetracycline-dependent promoters are engineered by placing tet...blog Plasmid 101: Inducible Promoters Plasmid 101: Repressible Promoters CRISPR 101: Which Cas9 Do I...tet response elements upstream of a minimal promoter. These systems come in two varieties: tet-on and tet-off...the genome. Make sure to choose an appropriate promoter to express your cDNA, depending on the level of... of expression you would like. Check out our promoter blog if you need help choosing. Viral expression....     Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's Lentiviral... proteins, as many of them just need a special promoter or tag prior to introduction of the construct ...

  16. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    ...pcDNA3, comes from the pcDNA series. It has the CMV promoter for mammalian expression, a multiple cloning site...yeast expression vectors with various markers and promoters. If you need to visualize YGOI on a western blot...vectors carrying several fusion proteins. Pick your promoter, selection cassette and visualization vehicle ...fly genome and are under the Gal4 inducible UAS promoter. Other vectors can be expressed in fly culture...culture cells like S2 and carry the metallothionein promoter (e.g. pMT-puro). Worm vectors There are several... following the expression pattern of a defined promoter region, ectopically expressing YGOI in a pattern...flanking sequences matching the sequences upstream and downstream of the open reading frame (ORF) of interest...

  17. Recombinase-based State Machines Enable Order-dependent Logic in vivo

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    Blog Post
    ...inducible promoter (PLtetO), the TP901 gene downstream of the arabinose (Ara)-inducible promoter (PBAD),...and promoters with buffer sequences on their 5’ and 3’ ends (for insulation). A list of promoters and ...serine recombinase genes downstream of inducible promoters: the BxbI gene downstream of the anhydrotetracycline...and the A118 gene downstream of the diacetylphloroglucinol (DAPG)-inducible promoter (PPhlF).  Output...Depending on the desired application, the inducible promoters we used to drive recombinase expression may be...demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved onto a GRSM register to execute...in this paper (recognition sites, terminators, promoters, and genes) were built with flanking primer binding...

  18. Plasmids 101: Terminators and PolyA signals

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    Blog Post
    ...polyadenylation generally serves to promote transcript longevity in eukaryotes and promote transcript degradation ...poly(A) signal. While many studies focus on promoter strength as a determinant of gene expression levels...termination. In a previous post we discussed the promoter's role in the initiation step of gene transcription...rbGlob) include the sequence motif AAUAAA which promotes both polyadenylation and termination. Out of those...but Has the Potential to Reduce Viral Titer in a Promoter-Dependent Manner. Hum Gene Ther. 2008. PMID: 18627247... Resources on the Addgene Blog Learn how the Promoter Controls Gene Expression Learn about New Cloning...transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically ...

  19. Lentivirus Production

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    Protocol
    .... Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...total per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this...produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation...T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells that are below passage 15...

  20. DAP arrays for multiplex gene editing

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    Blog Post
    ...strategy, the need for lengthy promoters, such as U6 or EF1α promoters, has limited the number of gRNAs...tandemly assembled tRNA-gRNAs is placed downstream of a lengthy promoter, which is used to drive the expression...from human or plant sources) in both the strength as a promoter and the activity to release gRNAs from ...previous tRNA-gRNA arrays, that rely on an upstream lengthy promoter to express the gRNA array, DAP arrays ...expression of itself, without using any additional promoter, which is followed by tRNA processing to release...installing functional genes to study and potentially treat polygenic diseases. This could also enable more ...

  21. CRISPR 101: Multiplex Expression of gRNAs

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    Blog Post
    ...from its own promoter. But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs...gRNA target sequence downstream of the 7SK, human U6, mouse U6, or human H1 promoters. If you express fewer...can I express more than one gRNA from a single promoter using a plasmid like pX330? Unfortunately, the...of pX330, contains humanized wtCas9 and two U6 promoters. To use this plasmid, you simply order oligonucleotides...contain Type IIS restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent...contain Type IIS restriction sites that flank the promoter-gRNA constructs. When these plasmids are digested...in a polyT-termination sequence for each unused promoter. This step is necessary to generate all of the...

  22. Plasmids 101: Modular Cloning

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    Blog Post
    ... individual part, this category also includes promoters, UTRs, terminators, etc. Once selected, individual...one or more gene(s), and typically begin with a promoter and end with a terminator. A simple TU could be... be comprised of just three parts: promoter, gene, and terminator.     Fig 1. MoClo enables individual...three steps: Level 0: Cloning of basic parts (promoters, UTRs, genes, tags, etc.) into MoClo-compatible...maximize production potential. For example, the best promoter for a given peptide can easily be tested by generating...generating Level 1 TUs with unique promoters, and the ideal phosphatase (or any modifying enzyme) to pair...additional components such as selectable markers, promoters, enhancers, etc. MoClo is especially useful for...

  23. Synthetic Photobiology: Optogenetics for E. coli

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    Blog Post
    ...experimented with the promoter strength of various components, removing inducible promoters that could crosstalk...outcome is directed transcription from a given promoter. To harness the power of light, Christopher Voigt... activates the RR (OmpR) via phosphorylation, promoting transcription (see figure below). Subsequent exposure...activates the response regulator via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure...chromosomally. Leakiness was also an issue; some promoter activity persisted in the inactive state. The ..., Schmidl et al. condensed the system to two streamlined plasmids. The utility of these TCSs was limited...improvements, the constructs have been greatly streamlined; only two plasmids are required per system. Applications...

  24. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    ...of a generic promoter). A DIO opsin allows cell type specificity dependent on the promoter of the cre recombinase...neuronal specificity through promoter-directed expression alone is extremely limited. Therefore, highly ...communicated to me as one of these extremely high expressing promoters). Conduct a time course experiment...for sufficiency of your circuit of interest in promoting reward is a self-stimulation experiment wherein...developed many of these AAV constructs with various promoters and other genetic manipulations to help restrict...specific neuronal subtype. However, the length of promoter that can be used in an AAV construct is limited...highly expressing, generic neuronal promoters such as synapsin or camKIIα drive most of the constructs. As ...

  25. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...dependent promoters, Baojun Wang’s lab developed a system that can activate sigma-54 dependent promoters (Liu...activation of sigma-54 type promoters. The system was functional in activating promoters in Pseudomonas syringae...that utilizes phage recombination machinery to promote homologous recombination of linear DNA fragments...regulate gene expression were limited to inducible promoters or direct gene knockout. In contrast, CRISPR offers...systems, catalytically dead (dCas9) targeted to a promoter or gene body can repress transcription by physically...and can be more effective targeting either the promoter or the coding sequence and the template or non-template...to multiplex repression. Using a Tet responsive promoter to drive expression of a catalytically dead Cas9...

  26. Plasmids 101: E. coli Strains for Protein Expression

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    Blog Post
    ...hybrid promoter. Although the DE3/T7 RNAP system works well for most experiments, the lac promoter can “...expression from high-copy number plasmids and powerful promoters will greatly exceed that of any native host protein... accomodate high protein loads using inducible promoter systems (which will be discussed later). In addition...Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter (found on vectors such as pQE or similar), inducible...above, many expression plasmids utilize inducible promoters, which are 'inactive' until an inducer such as...the culture to make protein.  The DE3 lysogen/T7 promoter combination is the most popular induction system...available to transcribe the gene of interest from a T7 promoter on the plasmid. Many commercial strains carry ...

  27. 28 Hot Plasmid Technologies from 2015

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    Blog Post
    ...all chemically inducible promoters; 3) improving the strength of the promoters used; and 4) optimizing ...fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various pieces...sequences. Several pre-constructed promoter-selection marker or promoter-inducible expression related vectors...ideal promoter/location combination. However, other constructs which contain different promoters and/or...to study the human GM-CSF promoter and enhancer, a finely regulated promoter controlled by a mixture of...multiple copies of a transcription factor to a promoter and localization of a protein via the presence...are inserted between the truncated gene and the promoter to prevent leaky expression of this resistance...

  28. Plasmids 101: Expressing shRNAs for RNA Interference

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    Blog Post
    ...RNA polymerase III promoters, which transcribe small RNAs. The most common promoters are U6, H1, and 7SK...sequence Choosing the plasmid backbone Choosing the promoter Mature siRNAs typically range from 19–22 nucleotides...7SK. These are strong promoters with no tissue specificity, so in some cases they can lead to toxicity...plasmids are being designed using RNA polymerase II promoters. The design of these plasmids is meant to more...closely mimic pre-miRNAs, and the use of the pol II promoters allows for more flexibility in expression levels...integrated expression atlas of miRNAs and their promoters in human and mouse. Nature Biotechnology, 35(9...Targeting RNA with Cas13a (C2c2) Plasmids 101: The Promoter Region – Let's Go! Browse All Plasmids 101 Posts...

  29. Multiplex Genome Editing with CRISPR-Cpf1

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    Blog Post
    ...cleavage sequence, tRNAs, multiple individual promoters).  spCas9 and its gRNAs are also larger than their...and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed Lentiviral Expression..., and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection...expressed in 293 cells. This allows for a single promoter to drive expression of a crRNA array and doesn...arrays eliminates the need to include multiple promoters to drive crRNA expression and sequences that allow... Zetsche et al reverses the orientation of the promoter driving expression of the crRNA array (bolded ... Make sure to order 5’ phosphorylated oligos or treat with T4 PNK. Delivery options for Cpf1 multiplexing...

  30. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

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    Blog Post
    ...at cancer cell metabolism. Cancer cells promote oxidative stress leading to the degradation of one key .... Unfortunately, as a whole, microenvironments promote tumor durability instead of repressing them, making...caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle progression and enhances tumor...signaling, possibly inducing the secretion of growth promoting proteins, such as human growth factor, vascular...Fibroblasts, or CAFs. CAFs are altered fibroblasts and promote the development and metastasis of tumors through...different organs. This means that exosomes not only promote microenvironment’s malignancy but also incite different...tumor’s evasiveness, evolution, and response to treatment. Some microenvironment cells improve tumor’s durability...

  31. Your Top Requested Plasmid in 2016!

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    Blog Post
    ...most imporant is that, upstream of the Cbh promoter, this plasmid has a U6 promoter followed by BbsI restriction...SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified version...version of CAG promoter that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course...

  32. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...proteins (bEBPs) at a sequence upstream of the promoter called the upstream activating sequence (UAS). The...σ54-based transcription. Transcription from σ54 promoters requires binding of bacterial enhancer binding...gRNA complex which allows transcription from the promoter.  They found that the system is active in E. coli... and displays high dynamic range. However, the promoter in question must be σ54-dependent and the system...and further the study of natural σ54-dependent promoter systems. Find these CRISPR activation plasmids... reporter by inserting the NanoLuc luciferase downstream of the apoB gene. Using this reporter, the researchers...small-molecule probes to aid in the understanding and treatment of cardiovascular disease. Find the LipoGlo plasmids...

  33. AAV Molecular Tools

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    Collection
    ...Gradinaru 99120 pAAV-ihSyn1-tTA Inducible Synapsin promoter (ihSyn) Expression of the tet-off transactivator...pAAV-ihSyn1-DIO-tTA Cre-dependent and inducible Synapsin promoter (ihSyn) Cre-dependent expression of the tet-off...amplified tTA expression. 1 Viviana Gradinaru 117383 TRE-DIO-eYFP Cre-dependent and Tetracycline-inducible...

  34. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...plasmids used to express CRE under neuron-specific promoters (aCaMKII and hSyn). Created with BioRender.com...plasmids used to express Cre under neuron-specific promoters, including calmodulin-dependent kinase II alpha...excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized...expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability to induce CRISPRa... CRISPRa in primary rat neurons over other promoters, including ubiquitous ones such as EF-1α and CAG....consequently regulates neuronal physiology by promoting the development of neurons and synapses as well...cells to determine which transcription factors promote neuronal differentiation (Liu et al., 2018). The...

  35. Lentivirus Plasmids

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    Collection
    ...shRNA under the control of a tet-responsive H1 promoter. Didier Trono 11651 pLVUT-tTR-KRAB 3rd Inducible...11795 pLL3.7 3rd Expresses shRNA under mouse U6 promoter. CMV-EGFP reporter cassette is included to monitor...epigenetic silencing. Expresses shRNA under mouse U6 promoter. CMV-EGFP reporter cassette is included to monitor...with a chimeric 5’LTR. Any expression cassette (promoter and gene of interest) can be cloned into the plasmid.... Didier Trono 17619 EF.CMV.RFP 2nd EF-1 alpha promoter for transgene and CMV drives expression of RFP...41393 pCW57.1 3rd Inducible lentiviral expression, TRE-gateway, with puromycin selection. See Root Lab pLEX... GFP Puro (658-5) 3rd eGFP expression with CMV promoter and puromycin selection. See Campeau et al. (2009...

  36. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ... cauliflower mosaic virus 35S promoter (CaMV 35S) or ubiquitin promoters (Belhaj et al., 2013). Addgene...achieved by placing the gRNA downstream of a plant-specific RNA pol III promoter, such as AtU6, TaU6, OsU6...consistent between plants and other organisms, but the promoter used to drive gRNA expression is dependent upon... gRNA” backbones which contain a plant pol III promoter and gRNA scaffold sequence and allow researchers...is commonly driven by plant-derived RNA pol II promoters which regulate expression of longer RNAs (such...expression). Examples of commonly used RNA pol II promoters for Cas9 expression include the ubiquitously expressing...Summary While different in many of the specifics - promoters used, precise protein sequences or domains, and...

  37. Networking and Inspiring Young Scientists at Freshers Fairs

    Type
    Blog Post
    ...fairs are also an opportunity for the societies to promote their upcoming events and intro sessions, and give...Since 1996, Cambridge Biosoc’s goal has been to promote interest in the biological sciences amongst the...night. The society also visits high schools to promote chemistry degrees and provide advice to students... to support iGEM alumni-industry networking to promote the creation of start ups and biotech spin offs...Laureates, who speak about their work and current, trending topics in their area of research. The society ...

  38. Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

    Type
    Blog Post
    ...tandem with a single promoter, but only the gene directly following the promoter is expressed. Cre-mediated... farther downstream in the construct. As RFP and OFP are directly downstream of the promoter in Brainbow...an RFP, CFP, and YFP downstream of a ubiquitous promoter. Zebrabow is an extremely versatile technology...to neurons by placing Brainbow downstream of a neural-specific promoter, or the entire fish could be labeled...Brainbow system are that it does not use ubiquitous promoters, meaning that not all cell types can be labeled...non-fluorescent YFP and activate fluorescence of the downstream OFP, GFP, or RFP. Cells that express the construct...adult fly. As a stop cassette is located directly upstream of the fluorescent protein genes, Cre recombination...

  39. Plasmids 101: Restriction Cloning

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    Blog Post
    ... the start of the gene to end up just downstream of the promoter found in the backbone. Ideally, the backbone... enzyme cut sites (restriction sites) downstream of the promoter as part of a multiple cloning site (MCS...place your gene in the proper orientation downstream of the promoter, you can add an EcoRI site just 5’ of... leaving the 5’ end of your gene just downstream of the promoter and placing the gene in the proper orientation...easily orient your gene insert with respect to the promoter. For example, let’s say your plasmid backbone ... on the left side of the image below. It has a promoter (blue arrow) followed by the restriction sites...of the backbone plasmid (see below). You should treat your digested backbone plasmid with a phosphatase...

  40. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

    Type
    Blog Post
    ...putting a gene of interest directly downstream of a tissue-specific promoter sounds simpler, this modular system...Gal4 expression can be regulated by any chosen promoter, and Bloomington’s Stock Center has fly lines ...lines expressing Gal4 under a variety of promoters resulting in tissue specific expression. To express YFG... to making a new fly with hedgehog under a new promoter each time a new hypothesis is tested. Controlling...gene expression. Thus, genes and siRNA encoded downstream of the UAS sequence are only expressed when Gal4...systems in Drosophila: A synthesis of time and space, Trends Genet. 20, 384–391 (2004). PubMed PMID: 15262411...I. Bartos, S. Marka, J. C. Kagan, Environmental Stress Causes Lethal Neuro-Trauma during Asymptomatic ...

  41. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will... proteins from the same mRNA. We've discussed promoters before so in this blog post we’ll cover basics...screenable or selectable markers from a unique promoter, multicistronic plasmids ensure that any cells...originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic ... the upstream cistron will have a few additional residues added to the end, while the downstream cistron...scientists have reported lower expression of the downstream cistron due to factors such as the experimental...the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and...

  42. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

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    Blog Post
    ...approximately 19 kb in length. It contains short promoter sequences at its 3’ and 5’ ends that are crucial..., the roles of specific viral sequences (e.g. promoters) can be studied by changing the minigenome cis-acting...minigenome) are under control of a T7 RNA polymerase promoter sequence. Thus, T7 RNA polymerase also needs to...leader region acts as a functional BRSV genome promoter." Journal of virology 73.1 (1999): 251-259. PubMed...Michael, et al. "The Ebola virus genomic replication promoter is bipartite and follows the rule of six." Journal...one developed for Zaire ebola virus (EBOV) are extremely useful, allowing researchers to study aspects ... systems, such as the one described here, are extremely useful research tools. In addition to the reduced...

  43. Stem Cell Models for Disease & Open Science: Interview with Darrell Kotton

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    Blog Post
    ...vitro models for lung disease. Darrell strives to promote open source biology in his own lab, at the Center...to help keep us focused on our core mission to promote scientific sharing. Listen to learn more about ... defines Open Science and how he practices and promotes open science in the lab - sharing reagents, cell...Darrell's ultimate goal - to develop therapies to treat disease in the lung. Chapter 2: Balancing research...Meidince - Understanding and developing means to treat disease through tissue repair, stem cells, gene ...

  44. Hot Plasmids - September 2019 - CRISPR, Auxin Inducible Degradation, MerMAID Channelrhodopsins, and Genome Looping

    Type
    Blog Post
    ...TIR1 of the SCF-TIR1 ubiquitin ligase complex, promoting its interaction with the degron sequence and ultimately...studying the effects of endogenous looping on distal promoters and enhancers, or have attempted to engineer loops...LADL Anchors to genomic targets, such as a distal promoter and an enhancer. What makes LADL unique from previous...group targeted the enhancer region of Klf4 and the promoter region of Zfp462, which are over 500kB apart, ...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...

  45. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ... gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene’s fly library web page... over 30 promoters and 140 genes, including constructs with unique tissue-specific promoters expressing...dCAS9-VPR expression is under the control of the TRE promoter, which allows for doxycycline-inducible expression...are cloned downstream of a minimal promoter sequence. Should one of these random stretches of DNA contain...its own transcription from the minimal promoter and its strength can be determined from its enrichment ...many different promoters, RBS’, coding sequences (CDS), and terminators of varying strengths. These can be...fluorophores, generate expression vectors with various promoters suitable for in vivo expression, and/or produce...

  46. Gibson Assembly Protocol

    Type
    Protocol
    ...technique is especially useful for introducing promoters, terminators, and other short sequences into the...creating a contiguous DNA fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein...

  47. Fluorescence Titering Assay

    Type
    Protocol
    .... Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for 293T...fluorescent cells, these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

  48. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    ...cell-type specific promoters; however, those usually drive weak expression of downstream genes. Strong cell-specific...Optogenetic FLEx vectors contain a strong promoter upstream of an opsin gene (e.g., channelrhodopsin-2...cell-specific promoters, on the other side, are too long for rAAVs, which can only package sequences shorter... the antisense orientation with respect to the promoter to prevent its expression, and is flanked (“floxed...rAAVs expressing Cre under a specific cell-type promoter. Once injected into the brain, the viruses infect...thereby enabling its expression under a strong promoter only in specific neuronal cells. FLEx ensures,...for analyzing the neural circuits of behavior." Trends in cognitive sciences 15.12 (2011): 592-600. PubMed...

  49. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...transcription start site, splice sites, exons, and/or promoters to knock out lncRNA function (Hazan and Bester...of introns or the removal of exons. Disrupting promoter or transcription start site function would also...effective in reducing off-target effects since promoter and enhancer sequences remain intact (Engreitz...Local regulation of gene expression by lncRNA promoters, transcription, and splicing. Nature. 2016;539...elements that are crucial for transcription and downstream function. Rather than attempting to cut out the...For example, inserting a polyadenylation signal downstream of the transcription start site will terminate... ncRNAs While CRISPR/Cas9 has proven to be an extremely useful system for modifying and assessing ncRNA...

  50. "Hall of Fame" AAV Enhancers from the Allen Institute for Brain Science

    Type
    Blog Post
    ...Plasmids 101: Gene, enhancer, and promoter trapping Plasmids 101: The Promoter Region – Let's Go! Plasmids...mediator complexes and the RNA polymerase at the promoter region of a gene to initiate transcription (Figure...cloned into an AAV backbone containing a minimal promoter to drive expression of a fluorescent gene and ...Transcriptional enhancers and their communication with gene promoters. Cellular and Molecular Life Sciences, 78(19–20...Science (AIBS). Scientists at AIBS have allocated tremendous time and resources to generate highly specific...describing the labeled regions, as well as the strength and density of labeling in each. This allows users...
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