...bacterial populations, an antibiotic resistance gene (i.e., a gene whose product confers resistance to ampicillin...transcription. Gene Knock-down Plasmids - Used for reducing the expression of an endogenous gene. This is frequently...chromosomes down to study their genes, marking the birth of molecular genetics. Today, scientists can easily...easily study and manipulate genes and other genetic elements using specifically engineered plasmids, commonly...when a gene is inserted within the MCS, its expression will be driven by the promoter. As a general rule...the gene, promoter, or other DNA fragment cloned into the MCS. The insert is typically the genetic element...experiment, see Addgene's empty backbone page for more information. Expression Plasmids - Used for gene expression...

...requires a helper plasmid containing genes from adenovirus. These genes (E4, E2a and VA) mediate AAV replication...adenovirus gene E1+, to produce infectious AAV particles. Rep/Cap and the adenovirus helper genes may also...Publications Viral serotypes and pseudotyping/gene therapy: Designer gene delivery vectors: molecular engineering...AAV) as novel vectors for human gene therapy. Grimm D, Kay MA. Curr Gene Ther. 2003 Aug;3(4):281-304. PubMed...affect the expression of transgenes, as well as the expression of neighboring genes. The short coding sequences...channelrhodopsins, halorhodopsins, and other optogenetic genes enables them to be packaged in AAVs. Click...aureus (SaCas9) . AAV is also a promising method for gene therapy. Of the commonly used viruses, AAV produces...

...knockin. Select gene and genetic element to be manipulated In order to manipulate a given gene using CRISPR...mutant allele of a gene ( point mutant )? Increase or decrease expression of a target gene? Once you have ...sequence for the gene you are trying to target. However, the exact region of the gene you target will ...want to: Generate complete and permanent loss of gene expression or function ( knockout )? Generate a specific... Edit Generate a specific user-defined sequence change in a particular gene, such as generating a point...reporter gene (e.g. GFP) to identify and enrich positive cells, or selection marker to generate stable ...promoter driving expression of your gene of interest. For genetic knockouts, gRNAs commonly target 5′ ...

...complex systems of gene regulation, like operons. Operons are a cluster of different genes that are controlled...transcription of genes. There are three types of RNA polymerases that all transcribe different genes. RNA polymerase...promoter is a region of DNA where transcription of a gene is initiated. Promoters are a vital component of...region controls when and where in the organism your gene of interest is expressed. Summary Promoters are ...of the sense or coding strand of the transcribed gene. The coding strand is the DNA strand that encodes...transcription. The most 3' portion (closest to the gene's start codon) of the core promoter is the TSS which...DNA sequences can affect transcription of a given gene. The TATA-binding protein binds the TATA box and...

...target gene. Positive selection screens are generally very robust, and tens of thousands of genes that ...contains a unique gene targeting sequence, but there are multiple sequences targeting each gene in the overall... multiple genes at once. A well-designed screen can help you begin to understand what genes are important...powerful tool for forward genetic screening, or identifying previously unknown genes that contribute to a ...list of genes involved in the selection NEGATIVE screen: Apply library Perform Next Generation Sequencing...identify genes that are essential for growth/survival under certain conditions. Next-Generation Sequencing... backbone, but they express or target different genes. Some libraries cover the majority of the genome...

...In lacZ gene M13 (-40) GTTTTCCCAGTCACGAC In lacZ gene M13 Reverse CAGGAAACAGCTATGAC In lacZ gene M13/pUC...Invitrogen) In lacZ gene M13/pUC Reverse AGCGGATAACAATTTCACACAGG (Invitrogen) In lacZ gene MBP-F GATGAAGCCCTGAAAGACGCGCAG...resistance gene, forward primer Neo-R GCCCAGTCATAGCCGAATAG 5' end of neomycin resistance gene, reverse ...reverse primer M13 Reverse CAGGAAACAGCTATGAC In lacZ gene MSCV CCCTTGAACCTCCTCGTTCGACC (BD Biosciences) Murine...ATAATACCGCGCCACATAGC 5' end of ampicillin resistance gene, reverse primer AUG1 Forward CAATTTACATCTTTATTTATTAACG...GCAACTGACTGAAATGCCTC 5' end of chloramphenicol resistance gene, reverse primer CMV Forward CGCAAATGGGCGGTAGGCGTG...GGGTTTTATTAACTTACAT (Vosshall lab) 5' end of Drosophila mini-white gene, reverse primer pCasper-hs GCAACTACTGAAATCTGCCAAG...

...Available online: Genetic Organization and Genetic & Structural Features . In vivo gene delivery and stable...transfer plasmid. 2nd generation lentiviral plasmids utilize the viral LTR promoter for gene expression, whereas... rev, and tat genes all from a single packaging plasmid such as psPAX2 . 3rd generation packaging systems...packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. The transfer plasmid contains the viral LTRs and...pictured). Unless an internal promoter is provided, gene expression is driven by the 5'LTR, which is a weak...: Uses 3 separate plasmids encoding various HIV genes. Safer. Replication incompetent and always SIN: ...standard retroviruses use the gag, pol, and env genes for packaging; however, they are different viruses...

...the early genes necessary for replication have been deleted from the shuttle vector. Early gene E1 is provided...genome are i nverted t erminal r epeats (ITRs). Genes are divided into early (E1-4) and late (L1-5) transcripts... to enter cells. Recombinant adenovirus has two genes deleted: E1 and E3. E1 is supplied by the adenovirus...33Kb adenoviral plasmid containing the adenoviral genes necessary for virus production. The shuttle vector...adenoviral vector. pAdEasy-2 does not contain the viral gene E4, adding 2.7 Kb of packaging space. However, these...however, AAV requires the presence of adenoviral genes E1, E4, E2a and VA for replication. For more information...event between your adenoviral vector and the early genes found in the packaging cell lines creates a wildtype...

... editing multiple genes at once; using dual nickases to generate a knockout or gene edit; or using Cas9...the targeted gene. The ideal result is a loss-of-function mutation within the targeted gene. However, the...eliminate target gene function. It has also been used extensively to screen for novel genes that regulate... activate, or repress genes. Each library typically contains ∼3–6 gRNAs per gene to ensure modification...repress, visualize, and isolate genes. Activation or Repression of Target Genes Figure 9: Overview of CRISPRi...highest levels of single-gene activation (Figure 9D) In bacteria, activating gene expression is more difficult... of effective gene activators when fused with dCas9. Recently, synthetic CRISPR-Cas gene activators have...

...MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol): Gag is a structural precursor protein... protein, and Pol is a polymerase. Envelope gene (may be pseudotyped to alter infectivity): The use of...both lentiviruses and γ-retroviruses use the same gene products for packaging (i.e., Gag, Pol, and Env)...and/or Env, eliminating the need to deliver these genes in trans by specific plasmids. This method, therefore...standard γ-retroviruses use the Gag, Pol, and Env genes for packaging; however, they are different viruses...replication incompetent? Yes, as with lentiviruses, the genes required for γ-retroviral packaging and transduction...U3 region of the 3’ LTR and are primarily used in gene therapy research to reduce promoter interference...

...receptor to engineer a receptor-effector complex for gene therapy. J Biol Chem. 276:1596-6. PMID: 11402033...Guides Chemogenetics Guide Chemogenetics Guide Jump To: Chemogenetics Guide Early Chemogenetic Receptors...to. To find chemogenetics plasmids available through Addgene, check out our Chemogenetics Plasmid Collection...Collection . Figure 1: General overview of a chemogenetics experiment. Early Chemogenetic Receptors: RASSLs...Addgene's guide to using Chemogenetics plasmids in your lab for interrogation of neuronal activity. Science...opto-chemogenetic fusion proteins of a light-emitting luciferase and a light-sensing optogenetic element...biophysical properties. While LMOs provide chemogenetic access to optogenetic tools, they also retain the ability...

...pDusk Blue-light photoreceptor for light-controlled gene expression. These plasmids rely on the engineered... Science Guides Optogenetics Guide Optogenetics Guide Jump To: Optogenetics Guide Introduction Microbial...Collection . To find optogenetics plasmids available through Addgene, check out our Optogenetics Plasmid Collection...Addgene's guide to using Optogenetics plasmids in your lab, including explanations of microbial opsin...References Introduction The field of optogenetics integrates optics and genetic engineering to measure and manipulate...from Kir2.1 540 Plan Your Optogenetics Experiment When designing your optogenetics experiment, you’ll need...are some key factors to consider: Optogenetic excitation or optogenetic inhibition. First things first: ...

...start or need a handy reference, Addgene's eBooks are here to help!... Addgene's eBook Collection When you're...sure where to start or need a handy reference, Addgene's eBooks are here to help! 0 of 0 Selected Download...

... backbone, but they express or target different genes. Some libraries cover the majority of the genome...genome, while others are restricted to certain gene sets. Read More Viral Vectors Find educational guides ...interest. Read More Chemogenetics Chemogenetics is a technique that uses genetically engineered receptors... experiment. Read More Optogenetics Optogenetics integrates optics and genetic engineering to measure ...links to the guides below. Antibodies Chemogenetics CRISPR Optogenetics Pooled Libraries Viral Vectors Antibodies... Science Guides Addgene has created the following guides to help you start...processes. The tools and technologies developed for optogenetics research utilize light to detect, measure, and...

... your gene of interest one time into a donor plasmid (or acquire one that already has your gene into it...molecular cloning scientists can amplify and manipulate genes of interest and then insert them into plasmids for...digests on our website. Restriction cloning of your gene of interest (YGOI) into a recipient plasmid. (Image...Addgene's reference guide on molecular cloning techniques, including information on seven commonly used...exonuclease digests the 5' end of dsDNA fragments to generate 3' single-stranded overhangs. DNA polymerases ... chews back the 5’ ends of your DNA fragments generating long overhangs that anneal to each other due ...to assemble before you get started. Check out Addgene's website for easy to use MoClo and Golden Gate ...

...Recombinant antibodies are created by cloning antibody genes from specific hybridoma cells into plasmids. Plasmids... tubulin (sometimes referred to as housekeeping genes). This allows for relative comparison of expression...Science Guides Antibody Guide Antibody Guide View Addgene's Antibody Collection Jump To: Antibody Guide Production...with myeloma cells (immortalized B cells). The genetic reorganization that creates variety in the antibody... be produced from plasmids, with an antibody’s genetic code inserted into the plasmid. This produces the...antibody is the antigen binding site. In B cells, the genetic region responsible for the variable region goes...epitopes on one antigen. Monoclonal antibodies are generated from cultured B cells, where each culture should...

...content on subjects such as: CRISPR Chemogenetics Optogenetics Genetic Code Expansion...Overview of Addgene's educational resources, including eBooks, science guides, videos, and protocols ... content , including protocols, tips on using Addgene’s materials, and career advice. Playlists include...Protocols Careers How-to Blog Read about plasmids in Addgene’s repository , and related molecular biology techniques...Concentrations for Bacterial Selection What is a Plasmid? Genetic Code Strain Information Molecular Cloning Techniques...