...showed the expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed...based on the ability to correctly assemble a lacI/lacZ cassette (designed by NEB for use in optimization...coding sequence for beta-galactosidase in the lacI/lacZ cassette. Additional confirmation of accurate assemblies...blue phenotype. Indeed, this was seen in all lacI/ lacZ assembly test systems; no blue colonies were obtained...from 1-, 12- and 24-fragment assemblies of the lacI/lacZ cassette, and illustrates how the volume of the ...fidelity and assembly efficiency Five fragment lacI/lacZ cassette assembly was easily achievable with high...both the 12- and 24-fragment versions of the lacI/lacZ cassette were designed and synthesized. In conjunction...

...the blue-white screen, which relies on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which can...on vectors where the gene of interest is inserted into the middle of the lacZ gene, thus disrupting 𝛽-... for blue-white screening at Addgene! Some bacterial strains contain lacZ in their genome, and scientists...where only cells that have gained or lost a specific gene survive (ex: antibiotic resistance marker). In a...scientists have found that when a portion of the gene is deleted, it creates a non-functional 𝛽-galactosidase...mentioned above. In molecular biology, cloning a gene into your plasmid is often a numbers game. By using...troubleshooting Sanger sequencing results. At Addgene, we now use next-generation sequencing during our QC process....

...specific genes are signified with a minus sign (-) as is typically standard; just having the gene listed... of the more common genetic changes found in E. coli strains. Table 1: Common gene mutations found in ...expressing the ccdB gene (important in Gateway cloning). F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1...lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14-  Φ80dlacZ∆M15 recA1...carefully selected the optimal promoter for your gene of interest, painstakingly cloned into the perfect...backbone, made sure to add the right tags to your gene, and may have even put a fluorescent protein downstream...in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase mutation (GATC...

...that deleting a section from the lacZ gene (a mutation called lacZΔM15) creates a non-functional β-galactosidase...well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme β-galactosidase...the plasmid containing the lethal gene. An example is the ccdB gene system commonly used in Gateway® Cloning...are rescued by genes supplied on the transformed plasmid. Such plasmids may contain genes that allow for...auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources on the Addgene Blog Read Our Post...other methods. Positive selection vectors encode a gene which, when expressed, is lethal to the cell. Cloning...fragments are inserted into an MCS in the center of this gene, disrupting the lethality. This is similar to α-...

...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...These vectors allow you to test the function of Your Gene Of Interest (YGOI) in a controlled environment under...design. You can create a new strain with a disrupted gene by transforming wild-type yeast with an integrating...involve using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following the expression...modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check... More Plasmid Resources from Addgene: Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other... is which vector will best suit your needs? At Addgene, we have a vast collection of empty backbones that...

...for the gene on the toehold switch to be expressed. In this case, the gene encodes the LacZ𝛼 protein....protein. Once expressed, LacZ𝛼 complexes with LacZ𝜔 and forms an active LacZ tetramer.  This tetramer cleaves...interfere with other genetic pathways making it difficult to link a phenotype to the gene being targeted via...need for mutant genetic backgrounds. The standard RNAi vector, L4440, places a target gene between two inverted..., highly efficient RNAi construct for C. elegans gene knockdown Article contributed by Alyssa Cecchetelli...extensively used in C. elegans research to study gene function. RNAi is commonly achieved by feeding E... from each promoter progresses past the specific gene producing long unspecific RNA fragments that are...

...Plasmids for endogenous gene tagging in human cells Resources on Addgene.org Browse all CRISPR plasmids...expressing a control -- for example, an FKBP12F36V-tagged LACZ) with dTAG molecules in your biological assay of...endogenous ENL was inactivated using CRISPR/Cas9 gene-editing. Featured molecules: dTAG-7 and dTAG-13 ...endogenous MELK was inactivated using CRISPR/Cas9 gene-editing. Featured molecules: dTAG-7, dTAG-13, and...Figure 1). Plasmids: We generated a series of plasmids that are available from Addgene. These plasmids enable...47 Additional Resources on the Addgene Blog Easi-CRISPR: Generating Knock-In and Conditional Mouse Models.... To overcome this limitation, we created a generalizable tag-based strategy, which we refer to as the...

...activation sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless...promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...Gal4 activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including...Shapira S.K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and ...hybrid system (also called the interaction trap), genes encoding proteins of interest(POI’s) are fused to... in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing the target... the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s interact...

...the ccdB gene is replaced by the gene of interest. As a result, cells that retain the ccdB gene die off...tool. The plasmid pKSCTMF contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR...antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of molecular...associated genes. These systems better ensure that plasmids are passed down through generations (Figure ...application of CcdB is GatewayTM cloning, where the ccdB gene serves as a counterselectable marker in the destination...death. The team used this cassette to knock out genes, knock in a GFP cassette, and introduced site-directed...toxin-antitoxin system to construct a stable synthetic gene circuit that can be used over prolonged periods....

...genome; does not restrict methylated DNA fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS...IPTG. T7 RNAP is then available to transcribe the gene of interest from a T7 promoter on the plasmid. Many...cytosine within a particular sequence. Browse Addgene's curated list of Bacterial Expression Systems Table...DE3) F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) General protein expression BL21 (DE3) pLysS* Chloramphenicol...ompT lon hsdSB (rB- mB-) gal dcm rne131 (DE3) General expression; not recommended for toxic proteins ...lon hsdSB (rB- mB-) gal dcm araB::T7RNAP-tetA General protein expression  BLR (DE3) Tetracycline RecA-deficient...-TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression  m15 pREP4*, ** Kanamycin (pREP4) Cis-repression...

...approaches increase the size of the gene delivered by splitting the gene into two pieces: part A and part...packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. ...how do you fit a big gene into a tiny vector like AAV? By breaking the transgene into smaller pieces. ...acceptor sequence, the 3’ half of the transgene. The full length gene is reconstituted by either the overlapping...Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids. Human Gene Therapy...Chamberlain JS (2011) Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination ...retinal gene therapy Learn about the parts of an AAV transfer plasmid Resources on Addgene.org: Check...

... cassette to the gene of interest. The gene to be expressed is amplified using a gene-specific forward...sgRNA-targeted gene, resulting in increased activation of endogenous transcription of the target gene. The brilliant...of downstream genes. Nihongaki et al describe the targeted activation of endogenous genes and detail the...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant*...resistance gene (missing the first two codons). Two stop codons are inserted between the truncated gene and ... inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (...on target gene activation. The novel dCas9-VPR activator is capable of enhancing target gene expression...