...Pre-constructed pairs of TALEN plasmids targeting human genes involved in cancer and epigenetic regulation from...TALengineering Reagents TALEN Reagents for Human Genes Engineered TALENs for Endogenous Human Gene Targets...large series of engineered TALENs targeted to human genes involved in cancer and/or epigenetic regulation ...

...Pre-constructed pairs of TALEN plasmids targeting zebrafish genes....target sites within various endogenous zebrafish genes; many of these were generated as part of an NIH-...TGCCTGGCACAGGGGCTctccaccatgctggccAACCTGTTCTCAATGA kinesin-family-member-13a TAL3106 & TAL3107 TGTTCTCTGTTTGAGCGAgtctccacacagcagaGCGACAGTAACAGCTTCA...

...in Mammalian Cell Lines CRISPR: Protocol for Genomic Deletions in Mammalian Cell Lines Other Addgene Resources...loss-of-function studies of genes and genetic elements in mammalian cell lines. Protocol For referenced ...CRISPR/Cas9 Clones for Deletions and Clone Selection For suspension cells, transfer all clones to a single...Validation of Biallelic Deletion Clones In order to characterize obtained clones and validate a successful knockout...JoVE) about genomic deletions in mammalian cell lines using CRISPR/Cas9 CRISPR...Cas9 to create genomic deletions in mammalian cell lines. Along with the video, you can find the protocol...Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9. Bauer DE, Canver MC, Orkin SH. ...

...Fluorescent Protein Guide Empty Backbones Fluorescent Protein Guide: Empty Backbones More Fluorescent Protein...Addgene has assembled a collection of empty plasmid backbones with different fluorescent tags for you to create...generated with the help of Erik Snapp . Empty Backbones for Fluorescent Protein Fusions (Organized by ...Excitation and Emission maximum are listed in nm. Brightness is the product of exctinction coefficient and...Blue/UV Protein Excitation (nm) Emission (nm) Brightness pKa Maturation Structure Plasmids Sirius 355 ...Top Cyan Protein Excitation (nm) Emission (nm) Brightness pKa Maturation Structure Plasmids Aquamarine ...

...A guide to Addgene's empty vector backbones curated by species, epitope tags/fusion protein, selectable... Plasmid Collections Empty Backbones Choosing Your Perfect Plasmid Backbone Background...consider. Here is a guide to Addgene's empty vector backbones. For the most part, we will assume that you... Host Relevant Promoters Representative Empty Backbones Mammalian CMV, SV40, EF1a, CAG pSG5L Flag HA -... high expression of UAS-driven transgenes Worm unc-54, variety of worm gene promoters C... and Lehner Lab vectors - Tools for nematode transgenesis Insect / Baculovirus Polyhedrin pFastBac LIC...Protein Common uses Representative Empty Backbones Flag Epitope tag pcDNA3 Flag HA - ...

...Guide RNA Expression Plasmids for Endogenous Human Genes...CRISPR-Cas/RGN expression plasmids for Human genes You may also like... CRISPR Guide CRISPR Protocols gRNA...technology to modify four different endogenous human genes (Fu et al., Nat Biotechnol. 2013). The expression...

...RNA Expression Plasmids for Endogenous Zebrafish Genes...different sites in various endogenous zebrafish genes (Hwang & Fu et al., Nat Biotechnol. 2013). The expression...

...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1m in neurons Yulong Li 123308-AAVrg...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1m in neurons Yulong Li 123309-AAV9...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1h in neurons Yulong Li 123309-AAVrg...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1h in neurons Yulong Li 123310-AAV9...the genetically-encoded fluorescent norepinephrine(NE) control sensor GRAB_NEmut in neurons Yulong Li 123310...the genetically-encoded fluorescent norepinephrine(NE) control sensor GRAB_NEmut in neurons Yulong Li 125560...combination with Cre-dependent transgenic mouse lines or co-infection with Cre dependent viral vectors...

...Syn genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1m Norepinephrine sensor Li 123309...Syn genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1h Norepinephrine sensor Li 123310...control norepinephrine sensor (does not respond to NE) Norepinephrine sensor Li Optogenetics 20297 pAAV-EF1a-double...serotype to deliver Cre-dependent transgenes in Cre transgenic lines. As part of our standard viral production...nuclear dTomato Calcium sensor Ting 100853 pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40 Syn Cre-dependent jRGECO1a...jRGECO1a Calcium Sensor Kim , GENIE 100854 pAAV.Syn.NES-jRGECO1a.WPRE.SV40 Syn jRGECO1a Calcium Sensor Kim...

...knockout screening in human cells. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods . 2014 Aug;11(8):...contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming...CRISPR-mediated nucleic acid cleavage. We have harnessed the type II CRISPR nuclease system to facilitate...implemented in mammalian cells by co-expressing the S. pyogenes Cas9 (SpCas9) nuclease along with the guide RNA...for the transcriptional activation of endogenous genes. It consists of three components: A nucleolytically..., Nature 2015). The dual vector system uses S. pyogenes Cas9 (SpCas9), using one vector to express SpCas9...

...SF-iGluSnFr Glucose Sensors iGlucoSnFr Norepinephrine (NE) Sensors GRAB_NE Voltage Reporters Archon Voltron...iGlucoSnFr mRuby2 Constitutive 9 Looger Norepinephrine (NE) Sensors 123308 pAAV-hSyn-GRAB_NE1m Syn GRAB_NE1m... Griesbeck Calcium Sensor: jRCaMP1 100846 pAAV.CAG.Flex.NES-jRCaMP1a.WPRE.SV40 CAG jRCaMP1a none Cre dependent... dependent 1 Kim , GENIE 100848 pAAV.Syn.NES.jRCaMP1a.WPRE.SV40 Syn jRCaMP1a none Constitutive 1, 9 Kim...Kim , GENIE 100849 pAAV.CAG.Flex.NES-jRCaMP1b.WPRE.SV40 CAG jRCaMP1b none Cre dependent 1 Kim , GENIE ...GENIE 100850 pAAV.Syn.Flex.NES-jRCaMP1b.WPRE.SV40 Syn jRCaMP1b none Cre dependent 1 Kim , GENIE 100851 pAAV.Syn.NES-jRCaMP1b.WPRE.SV40...CaMPARI none Constitutive 1, 5, 9 Looger 101060 pAAV_hsyn_NES-his-CaMPARI2-WPRE-SV40 Syn CaMPARI2 his Constitutive...

...Bacterial TAG Ryan Mehl 174081 pAcBac1-NES-Flag-R2-84-MbRS Tetrazine3.0 NES-Flag-R2-84 tRNA synthetase M. barkeri...growth medium, ncAA concentration, and the cell lines used previously. Remember that the orthogonal pairs...aminoacylate only the orthogonal tRNA, and not endogenous ones. Endogenous synthetases cannot aminoacylate the ...Andrea Musacchio 182287 pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF) Y306A/Y384F (AF) pyrrolysine (Pyl) tRNA synthetase...Nikić-Spiegel 182652 pcDNA3.1(+)_4x(U6 tRNA M15)_CMV NESPylRS(AF) Y306A/Y384F (AF) pyrrolysine (Pyl) tRNA synthetase...Nikić-Spiegel 182653 pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF)_IRES_eRF1(E55D)-HA Y306A/Y384F (AF) pyrrolysine... 197099 pIYN3 TyrRS M. jannaschii halogenated tyrosines Bacterial TAG Kensaku Sakamoto 197100 pCDF-Mm2...

...pcDNA3.1-hSNCA-NE SNCA NE CMV Parkinson's Shu Leong Ho 102363 pcDNA3.1-hNurr1-NE NR4A2 NE CMV Parkinson's...pSIN-PAmCherry-mSNCA-NE SNCA NE EF-1 alpha Parkinson's Shu Leong Ho 102366 pSIN-hSNCA-NE SNCA NE EF-1 alpha Parkinson's...Alzheimer's Hélène Marie 107544 pAAV-AICD-NES-IRES-hrGFP APP V5 CMV Alzheimer's Hélène Marie 107548 pAAV-syn-AICD-IRES-hrGFP...pAAV-syn-AICD-IRES-hrGFP APP hSyn1 Alzheimer's Hélène Marie 107594 pCS2-sod1 SOD1 SP6 ALS Qing Deng 107798...SFFV Parkinson's Denes Hnisz 145270 pHR-TBP(53Q)-IDR-mCherry-Cry2-NLS TBP Parkinson's Denes Hnisz 145283 ...as fluorescent protein (FP) tags. Information on genes and associated neurodegenerative diseases was curated...145283 pET-45b-TBP(38Q)-IDR-mCherry TBP Parkinson's Denes Hnisz 146392 pAc5.1B-lambdaN-HA-DmeIF4G-trunc-V5His6...

...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8...Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus You may also...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated...transient transfection approaches, generating cell lines using lentiviral vectors enables long-term protein...293T cells but can be adapted to alternative cell lines. Last Updated: January 19, 2019 Workflow Timeline...needed Day 14-18: Expand and harvest stable cell lines Equipment Biosafety cabinet Pipetman Pipettors Incubator...

...AID) enzyme. AID, however, only changes cytidines or guanines to the other three bases, which limits the...directed mutagenesis to generate specific mutations in your plasmids Use REPLACER Mutagenesis to mutate...Mutagenesis is a tool that both evolution and molecular biologists use to tinker with DNA. Making changes...evolution of new phenotypes, and forward genetics harnesses this at a large scale by screening diverse libraries...region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods...error prone polymerase I (PolI3M) is capable of mutagenesis in E. Coli, but this approach requires special...

... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...Laursen from Cornell University. Site directed mutagenesis is a highly versatile technique that can be used...analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and...relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate...plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG...Cas9 induced cleavage. Summary of Site Directed Mutagenesis In brief, point-mutations can be introduced to...the desired modification. Finally, the positive clones are sequenced to confirm the desired modification...

...practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene depositor...with Venus. This luminescent technology is based on the principle of bioluminescence resonance energy ...reporter genes for endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent signal... a few drawbacks associated with luminescent imaging: A luminescent substrate is required for imaging...Researchers have long been interested in using luminescence to get around these issues, but this solution...fusion proteins. Solving the problems of low luminescence By itself, Renilla luciferase has a low quantum... hasn’t stopped some scientists from using chemiluminescent proteins for imaging, but it has precluded...

...allowed scientists to tag and light up endogenous genes of interest to better understand normal protein ...vectors. These vectors can be used to tag endogenous genes and simultaneously introduce a selection marker ...vectors can be easily modified to tag hundreds of genes, generating powerful genetic screening libraries...easily create C-terminal tag fusions of endogenous genes in human cells. To use CRISPaint, users need 3 vectors... Learn more about these constructs and the cell lines they’ve been used to create in the Allen Institute...

...expression of surrounding genes. However, there are ways to create scarless mutations, ones that don’t leave any...toxins for counter-selection In 2015, the Chen lab harnessed a group of bacterial toxins to facilitate efficient...Red that contained one of eight bacterial toxin genes isolated from E. coli or Psuedomonas aeruginosa....toxins, and phage holins. They hypothesized that genes that had evolved specifically for toxicity would... pathogenic Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible promoter, ... they are just spontaneous antibiotic resistant clones. But if they are blue or magenta, they are real...

...would spend hours to days trying find their correct clones. Additionally, the presence of a plasmid is disadvantageous...Historically, antibiotics have also been used to disrupt genes at the chromosomal level. Scientists introduce an...