...the TRE, permitting transcription. Tetracycline-dependent promoters are developed by placing a TRE upstream...third-generation rtTA On Elledge 19407 pTREtight2 Promoter contains a modified TRE that is silent in absence of ...inducible promoters capable of controlling eukaryotic gene expression. Several endogenous promoters had been...plasmids. Tet System and the Tet Response Element (TRE) A TRE is seven repeats of a 19 nucleotide tetracycline...from a bidirectional tet-responsive promoter (Pbi); Pbi contains a TRE between two minimal CMV and is silent....CMV.tTA Adenoviral; CMV promoter; see article for neuronal-specific promoter and enhancer options tTA...

... 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...

... 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 ...

... 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...

...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...

...Stainier 85040 pK170.AAV-TRE-Cre-WPRE (Supernova) TRE-Cre based Supernova TRE AAV Iwasato 85577 pTC-...system is tight temporal regulation. Promoter-regulated Cre: The promoter region defines the areas in which...active promoter like CAG, or expressed only in a subset of cells under a more specific promoter (e.g. ... PBAD promoter Bacterial Richmond 112614 pVHC Venus and Cre-ERT2 with MCS for inserting promoter none ...inserting promoter none Mammalian Heller 112616 pmTHC TFP and Cre-ERT2 with MCS for inserting promoter none...GFP and Cre CMV Lentiviral Lu 118029 pAAV-TRE-dDIO-vCre VCre TRE AAV Luo 119063 pBac(3xP3-gTc’v; Tc-hsp68...CAG Mammalian Zhang 68477 pTRE:iRFP670-EFS:Cre-2A-GFP iRFP670, Cre, and GFP TRE Mammalian Jacks 68544 AAV-Cre-GFP...

...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...

...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...

...controlled by SNR 52 promoter, targeting endogenous TRE elements of pTET07 promoter...retroVirus LTR promoter. The second plasmid ( pgRNA_humanized ) contains a murine U6 promoter controlled ...vector containing murine U6 promoter and sgRNA targeting GAL4 UAS promoter 46916 pU6-sgGAL4-4 Human pSico-based...vector containing murine U6 promoter and sgRNA targeting GAL4 UAS promoter (negative control) 46918 pU6...contains sgRNA controlled by SNR 52 promoter, targeting endogenous TEF1 promoter 46923 pSNR52-sgTET Yeast CEN...pHR-SFFV-dCas9-BFP Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS and tagBFP 46911...BFP-KRAB Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS, tagBFP and a KRAB...

...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...

...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...for amplified tTA expression. 1 Gradinaru 117383 TRE-DIO-eYFP Cre-dependent and Tetracycline-inducible...

...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 ...

...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...

... 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...

...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...

...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...

...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 ...

...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...

...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...

...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 ...

...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 ...

...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...

... 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...

...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...

...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 ...

...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...

...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 ...

.... 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...

...shRNA under the control of a tet-responsive H1 promoter Trono 11651 pLVUT-tTR-KRAB 3rd Inducible expression...11795 pLL3.7 3rd Expresses shRNA under mouse U6 promoter; CMV-EGFP reporter cassette is included to monitor...silencing were added; 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...coexpression. 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; PGK-rtTA-2A-puro. See article for more versions...

...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...

...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...

...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...

...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...

...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...

...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...

... 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...

...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 ...

...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...

... 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...

...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 ...

...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...

...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...

...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 ...

...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...

... 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...

...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...

...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...

... Pro-Tips 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...

...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...

... elements are cloned into a plasmid upstream of a minimal promoter linked to luciferase. Activation of...enzymatic activity is measured. The effect of promoters or enhancer regions on gene expression can be ...investigate the effect of regulatory elements, such as promoters, enhancers and untranslated regions, or the effect...Cofactors/Substrates Required Not Required Signal Strength Lower Higher Sensitivity Higher Lower Background...regulatory element (wild-type or mutant) is cloned upstream of the luciferase gene in the plasmid, which is...Expression of luciferase can be monitored as a downstream measurement of miRNA efficiency on a target sequence...The gene of interest is cloned into a plasmid downstream of luciferase containing a stop codon, which ...