...Lentiviral BsmBI yes, cut S. pyogenes EGFP Ebert pLKO.1-puro U6 sgRNA BfuAI stuffer 50920 Mammalian/Lentiviral...Worm HindIII none S. pyogenes de Bono sgRNA with rpr-1 promoter 48961 Worm EcoRI none S. pyogenes de Bono...Zebrafish BseRI none S. pyogenes Zon pCRISPomyces-1 61736 Bacteria BbsI yes, cut S. pyogenes Apramycin...see plasmid page yes, cut S. pyogenes BFP Kuhn pLKO.1-puro U6 sgRNA BfuAI large stuffer 52628 Mammalian/...Puro Zoldos pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem(1/110) 71707 Mammalian hU6 yes, cut S. pyogenes Draetta...expression of Cas9, dCas9, or dCas9-VP64 along with 1-4 sgRNAs expressed from independent promoters. sgRNAs...for the expression of two gRNAs from Drosophila U6:1 and U6:3 promoters. Bullock pCFD5 Drosophila Utilizes...

...phage display. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. Carlos Barbas Vector system for expression...immunostaining. Biochem Biophys Res Commun. 2015 Jan 2;456(1):527-33. Gavin Wright pET-30-based vector dedicated...mAb Collection . Once the plasmids are transiently transfected into mammalian tissue culture cells, the...making synthetic nanobodies (sybodies) against membrane protein targets. Synthetic single domain antibodies...antibodies for the conformational trapping of membrane proteins. Elife. 2018 May 24;7. and Generation of...

...populations defined by multiple parameters. Figure 1: Examples of intersectional cell population definitions... as a proof-of-concept targeting approach in 2014 1 (using EYFP and ChR2-EYFP as payloads). This approach...interneurons encode fear memory. Nat Neurosci. 23(1):61-74. PubMed (Link opens in a new window) Hafner...Itch-Scratching Cycle via Descending Regulation. Neuron 101(1):45-59. PubMed (Link opens in a new window) Lazaridis...including dual-transgenic recombinase-expressing mouse lines 2–9 and combinations of transgenic recombinase-expressing... Cre AND Flp, here expressed through a double transgenic animal strategy (PV-2a-Cre; SOM-IRES-Flp). Image...in a new window) of Flp-recombinase-expressing transgenic mouse and rat lines. The Fenno Lab maintains ...

...handled at BSL-1, AAVs expressing oncogenes or toxins should be handled at BSL-2. AAV can transduce both dividing...Addgene ID Plasmid Capsid Description PI 112862 pAAV2/1 AAV1 AAV packaging plasmid, expressing Rep2 and Cap1...Addgene AAV transfer plasmids Viral... AAV does not integrate into the host genome, transgene expression can be long-lived. The utility of AAV...viral genome (typically called cis -plasmid or transfer plasmid). Helper Plasmid ID Plasmid Serotype Description...

... regulate a genome instead of modifying a genome. 1. Two-plasmid CRISPRi system for bacterial gene knockdown...repetitive sequence of human MUC4 exon 3 46915 pU6-sgGAL4-1 Human pSico-based U6 vector containing murine U6 promoter...efficiently block transcription (in bacteria, ~300-fold repression) by interfering with transcriptional elongation...for selection or fluorescent gating of transiently transfected cells. Co-expression of both plasmids in...elongation, RNA polymerase binding, or transcription factor binding. The binding specificity is determined jointly...the nuclease-deficient mutant could silence transcription on the gene expression level without genetically...CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Gilbert LA, Larson MH, Morsut...

...Bacterial TAG Farren Isaacs 73545 pEvol-pAcFRS.1.t1 pAcFRS.1.t1 E. coli p-acetyl-l-phenylalanine (pAcF) Bacterial...Bacterial TAG Farren Isaacs 73547 pEvol-pAzFRS.1.t1 pAzFRS.1.t1 E. coli p-azido-l-phenylalanine (pAzF) Bacterial...7FTrp) Bacterial TAG Thomas Huber 207639 pEVOL-NBK-1 PylRS M. mazei Pyrrolysine Bacterial Cole DeForest...and tyrU gene replaced by GentR) and release factor 1. Expresses archaeal MjTyrRS/tRNA pair instead. For...protein translation machinery. To expand the genetic code, 4 major changes to the standard translation machinery...Bacterial Ahmed Badran 209739 pAB228i24 G1 PylRS Methanogenic archaeon Bacterial Ahmed Badran 209740 pAB228q12...jannaschii Bacterial Ahmed Badran 209741 pAB228r3 Mlum1RS Bacterial Ahmed Badran 209742 pAB228p PylRS M. ...

...sequences containing a G nucleotide at position 20 (1 bp upstream of the PAM) may be more efficacious than...population. Some cells may be wild type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack... in vitro and in vivo. Nature Biotechnology . 33 (1), 73–80. PMID: 25357182...For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may...mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA can be present...present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene ...screens using CRISPR AAV transduction CRISPR elements are inserted into an AAV transfer vector and used to ...

...donor and acceptor. FRET is often used to study (1) protein-protein interactions where each protein is...standard consisting of ECFP and EYFP connected by 1 flexible glycine- and serine-containing peptide linker...fused to the N-terminus of LSSmOrange pLSSmOrange-C1 Orange Mammalian Express a gene of interest fused ...resonance energy transfer, or FRET , is a process by which energy is non-radiatively transferred from an excited... GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest fused...excited donor fluorophore to an acceptor. Since the transfer of energy does not occur by emission of a photon...FRET experiments. The efficiency of the energy transfer is measured using fluorescence microscopy by exciting...

...for any application TMN200 (200 mM sodium chloride, 1 mM magnesium chloride, 20 mM Tris at pH 8.0) Suitable...adeno-associated viral vector. Hum Gene Ther Methods . Feb;25(1):72-82. PMID: 24191859 Ye, et al. 2015. Safety and...enhanced transduction of the retina following subretinal administration and enhanced transduction and spread...cite Boye, et al. 2016. Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-... from the University of Florida for enhanced transduction of the eye and other tissues. As part of our...Y444F) serotype is demonstrated to have enhanced transduction of the retina following intravitreal injection... AAV2(Y444F) serotype also displays enhanced transduction of the trabecular meshwork following intracameral...

...Buenrostro JD, Regev A, Zhang F. Cell 2023 Jan 5; 186(1):209-229. PubMed (Link opens in a new window) . TF...Browse the Zhang lab’s comprehensive human transcription factor collection, available at Addgene. The...The collection can be used for screening transcription factors for cellular programming or cancer drug ...Library The MORF library is a comprehensive human transcription factor collection that contains 1,836 genes ...collection, can be purchased at Addgene. Each transcription factor isoform has a unique 24 bp barcode that...that simplifies identification of the transcription factor isoform in pooled screens. This collection ...collection and library can be used for screening transcription factors for applications such as cellular programming...

...in mammalian cells or zebrafish). pMG10 (a '“2-in-1” ZFN cassette for expression of both a “+” and “-”...different cell types. Zinc finger arrays can be transferred in-frame to these vectors. Plasmid...either OPEN or modular assembly can be easily transferred in-frame to any of these vectors using unique...

...Expression mEGFP 488 507 34 6 Monomer (A206K) pmEGFP-1 - Mammalian Expression mEGFP-N1 - Mammalian Expression...TurboRFP-pBAD - Bacterial Expression tdTomato 554 581 95 4.7 1 hr Tandem-dimer pCSCMV:tdTomato - Mammalian Expression...-pBAD - Bacterial Expression mRFP1 584 607 13 4.5 1 hr Monomer pcDNA3-mRFP - Mammalian Expression pMXs-mRFP1... of Biomedical Optics, Feburary 2005, Vol. 10 No. 1, p. 14003 Chu et al. : Nature Methods, May 2014 Vol... Monomer pET mOrange LIC cloning vector (u-mOrange) - Bacterial Expression pCS2+8CmOrange - Zebrafish/...pCS2+8NmOrange - Zebrafish/Xenopus/C.elegans/Sea urchin mOrange-N1 - Mammalian Expression mOrange-C1 -...Expression mOrange-pBAD - Bacterial Expression mOrange2 549 565 35 6.5 4.5 hr Monomer mOrange2-N1 - Mammalian...

...research and innovation programme and EFPIA. Figure 1: Representation of RESOLUTE mission. The combination...CRISPR knockout library targeting human transmembrane transporters. Feedback Please send your feedback to...carrier proteins (SLCs) are the largest family of transporters in the human genome with approximately 450 identified... Medicines Initiative 2 Joint Undertaking under grant agreement No 777372. This Joint Undertaking receives...allow a fast and efficient sub-cloning into a wide range of Gateway-compatible pDEST vectors. ID Plasmid ...overexpression library of human SLC proteins. Human Transporter Knockout Libraries 213695 221409 221410 Superti-Furga...

...Biology, 2(9), 537–541. DOI: 10.1021/sb400021j Figure 1: Multiplexing CRISPR knockdown to modulate expression...controlling the number of mismatches we can obtain a range of expression levels. This collection comprises ...Together, these plasmids allow to explore the whole range of expression levels for these two reporters simultaneously... S. (2018). Tuning dCas9’s ability to block transcription enables robust, noiseless knockdown of bacterial...

...gene activation CRISPR/Cas9 Plasmid Variants Figure 1: Schematic of the CRISPR Collection. Plasmids 159654... and 159655 are single guide expressing double stranded break constructs. Plasmids 171098 and 175570 allow...

...proteins in budding yeast. Sci Rep. 2019 Feb 19;9(1):2234. doi: 10.1038/s41598-019-38913-z. PubMed 30783202...Looger LL, von Wirén N, Frommer WB. A cytosolic trans-activation domain essential for ammonium uptake....

...) a T ranscriptional R epressor D omain (TRD) the TRD contains a Nuclear receptor Co-Repressor 1/Silencing...affecting females at an incidence of approximately 1:10,000. It is characterized by apparently normal development...reactivation, RNA editing, gene replacement therapy, RNA trans-splicing, and protein replacement to reverse the...cases Rett syndrome can be inherited. In females, random X inactivation results in approximately half of...disease severity. The MECP2 protein is a global transcriptional regulator of thousands of genes and studies...studies have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling...DNA and recruit known co-repressors or other transcriptional modifiers suggests the major function of MECP2...

... your account. We will notify you by email within 1 business day regarding your account verification. ... Material Transfer Agreements Most Addgene orders must be accompanied by a material transfer agreement...industry scientists. Please note this does not guarantee availability but lets us know what plasmids would...agreement (MTA), including transfers to for-profit entities. The MTA is a legal agreement between the recipient... the depositing institution(s). It governs the transfer of biological materials and intellectual property...Please see the Sample Agreements on our Technology Transfer Information page for more information. To learn...

...repression. The new transactivator rtTA ( r everse t etracycline-controlled t rans a ctivator) was created...CMV rtTA3 Blast (w756-1) Lentiviral reverse tetracycline-controlled transactivator 3 (rtTA3) expression...TetR On Geley Transactivators (tTA or rtTA) Find a construct that expresses the transactivator for your tetraycline...Overview of tetracycline regulated inducible transcriptional activation or repression Plasmid...tetracycline and not to the TRE, permitting transcription. Tetracycline-dependent promoters are developed... gene expression, a tetracycline-controlled transactivator (tTA) was developed. tTA was created by fusing... of VP16 (virion protein 16), an essential transcriptional activation domain from HSV (herpes simplex ...

... Caltech and can help guide your decision. Figure 1: Decision tree for choosing your AAV capsid. Your ...tdTomato Control Boyden 83900 pAAV-mDlx-GFP-Fishell-1 Dlx GFP Control Fishell 99130 pAAV-mDlx-NLS-mRuby2....AX with GRE or transgenic animal Oligodendrocytes - AAV-PHP.eB with GRE or transgenic animal Brain vascular....AX with GRE or transgenic animal L5 & Inhibitory - AAV-PHP.eB with GRE or transgenic animal L2/3 & L4...AAV.CAP-B10 with GRE or transgenic animal Excitatory - AAV.PHP.N with GRE or transgenic animal For PNS applications... pAAV-S5E2-GFP-fGFP E2 regulatory element GFP, membrane-targeted GFP Control Dimidschstein 192552 pAAV-CAG... viral prep format. Please note this does not guarantee viral vector packaging service, but lets us know...

...formulation buffer is phosphate-buffered saline containing 1 mM sodium azide. This buffer is not compatible for...extracted from cells, separated by SDS-PAGE, transferred to a membrane, and labeled with the antibody and a species-specific...expression vectors, human embryonic kidney cells are transfected with the plasmid(s) encoding the heavy and light...anti-GFP antibody), the target antigen is first transiently expressed in mammalian cells. To learn which ... The fluorescence pattern is compared to an untransfected control. Immunohistochemistry (IHC) Tissue sections...antibody. Protein expression is compared to an untransfected control and the protein size is confirmed. Residual...contain low levels of co-purified plasmid from transfection. This residual plasmid is extracted and amplified...

...cell surface proteins. J Am Chem Soc. 127(37):12770-1. PMID:16159249...with a recognizable sub-cellular localization (membrane, nucleus, etc.) are also available Non-toxic to... is based on the human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein, and is suitable...Vector SNAP-tag Empty backbone for stable and transient mammalian expression 101133 pSNAP-CaaX Control...Control Plasmid SNAP-tag Control for endomembrane (CaaX box) localization 101129 pSNAPf-Cox8A Control Plasmid...Vector CLIP-tag Empty backbone for stable and transient mammalian expression 101134 pCLIPf-H2B Control...pMCP-tag (m) Vector ACP/MCP-tag Empty backbone for transient mammalian expression 101126 pACP-tag (m)- 2 Vector...

...available as a substrate for another round. Figure 1: Overview of prime editing. Created with BioRender.com...incorporated into the DNA by reverse transcription. The edited DNA strand displaces the unedited 5’ flap and...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...replace” gene editing method in which a reverse transcriptase (RT) is fused to the C terminus of Cas9 H840A...the target sequence where it nicks the non-target strand and generates a 3’ flap. The 3’ flap binds to the...Moloney murine leukemia virus (M-MLV) reverse transcriptase. Engineering of the PE enzyme and related components...including exploring new sources for the reverse transcriptase. Many PE enzymes and related tools are available...

...component involved in GTPase signaling activity. Figure 1: Locations of ARF superfamily members. Figure from...regulators of membrane traffic, they also play critical roles at cilia, centrosomes, the plasma membrane, lipid...2016). Tracing the archaeal origins of Eukaryotic Membrane-Trafficking System building blocks. Molecular ...., Dacks, J. B., Lambright, D. G., Lee, F. S., Randazzo, P. A., Santy, L. C., Schürmann, A., Wilhelmi,...

...64325 3xFLAG-dCas9/p-bacteria 3xFLAG-dCas9 pLtetO-1 Fujii Efficient isolation of specific genomic regions...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...

...coming closer to this DNA targeting ideal. Figure 1: (A) Representation of a zinc finger (ZF) nuclease..., that will probably never happen. Yet with transcription activator-like (TAL) effectors, scientists in...sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol. 2011 Feb;29(2):149-53. PMID...

...material to specific cells in an organism. Figure 1. Using viral plasmids to generate virus. A) Viral ...Adenoviruses have high transduction efficiency and direct high but transient transgene expression. γ-Retrovirus...virus for gene transfer in clinical trials. These viruses have a relatively high transduction efficiency,...Dividing/Non-dividing Yes Low AAV Transient or Stable* single stranded linear DNA ∼4.5 Kb 18-26 Dividing.../Non-dividing No* Very Low Adenovirus Transient double stranded linear DNA >8 Kb 105 Dividing/Non-dividing...are versatile tools because of their ability to transduce non-dividing cells. These viruses enable long-...immunogencity. These viruses can direct long-term transgene expression, but generally are not permanently ...

...Hyg Chen 86196 pYPQ141-ZmUbi-RZ-As Maize ubiquitin 1 none As Cpf1 Qi 91715 pKEE401 yes, cut S. pyogenes...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...

...to determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...Link opens in a new window) . Transfections are performed using the transfer plasmid, a plasmid encoding...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...opens in a new window) ). Briefly, 293T cells are transduced with serial dilutions of a lentiviral vector,... taken off-line and discarded. Confirmation of Transfer Plasmid Addgene uses a rigorous barcode matching...

...one of three different inherited genes: Presenilin 1, Presenilin 2, and APP gene.The majority (>90%) of...used to transform iPSCs and fibroblasts into neurons and more. New and Noteworthy: Study aberrant axon initial...associated with the disease and implicate a wide range of cellular processes. The vast majority of cases...kinetics and localization for imaging synaptic transmission. Aggarwal et al. Nat Methods. 2023 May 4. See...PiggyBac plasmids with tet-inducible expression of transcription factors for sensory, motor, and cortical neuron...Use a FRET-based method to monitor structural transitions in protein self-organization. Wan et al. Cell...

...PDF 2.3 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9...and Wente Zebrafish: gRNA cloning, in vitro transcription, injection gRNA core ; Cas9 ; optimized Cas9...102.5 KB Jaenisch CRISPR plasmid summary for transcriptional activation dCas9 activators sgRNA dual expression...

...mediating the entry of SARS-CoV and SARS-CoV-2 viruses. ( 1 ) TMPRSS2 - a serine protease that primes the SARS-CoV... Plasmids expressing Cas12a/Cas13a, Reverse Transcriptases, etc. that may be useful for detection assays...site for furin proteases.( 3 ) BSG - (CD147), transmembrane glycoprotein of the immunoglobulin superfamily...proteolysis of SARS-CoV Spike protein, activating its membrane fusion function.( 5 ) TACE - (ADAM17) a metalloprotease... method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR). Mammoth Biosciences has published...2020.06.23.166397 (Link opens in a new window) reverse transcriptase, protease, and Bst polymerase constructs now...unpublished) Taq polymerase and MMLV reverse transcriptase expression plasmids from the Drew Endy and Philippa...

...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for...proteins by size. Transfer proteins from the gel to a membrane. Incubate the membrane with an unconjugated...block the membrane to remove excess antibody and prevent unwanted binding. Incubate the membrane with a ...Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....variable regions, shown in light blue and light orange, that bind to a specific antigen, triggering an...

...NLS-mRuby2 Control Gradinaru 83900 pAAV-mDlx-GFP-Fishell-1 Dlx GFP Control Fishell 83895 pAAV-hDlx-Flex-GFP-Fishell...this serotype to deliver Cre-dependent transgenes in Cre transgenic lines. As part of our standard viral...serotype permits robust retrograde access and transgene delivery to projection neurons. Specific classes...titering by SYBR green qPCR , confirmation of the transfer plasmid with PCR, purity assessment by silver ... viral prep format. Please note this does not guarantee viral vector packaging service, but lets us know...

... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...dynamic range, and resistance to photobleaching has made luciferase a common choice in assays ranging from... luciferase. Transcriptional Element Screening Optimized STARR-seq ( S elf- T ranscribing A ctive R egulatory...including transcriptional reporters for NF-kb, TGF-b, c-Myc, p53 and MAPK/JNK transcriptional reporters...collection of luciferase plasmids for assays of transcriptional regulation and bioluminescent reporters. Plasmid...bioluminescence found in a diverse number of organisms, ranging from bacteria to aquatic animals to insects. Luciferase... protein half-lives, making them useful as transcriptional reporters. However, they are not secreted, ...

...expression inducible by red light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline...testing transcription factor binding to reporter plasmid pH3U3-zif268 (omega) . Transcription factors ... answer many interesting questions. Does my transcription factor bind to a particular sequence? Do these...wild-type Cas9 from S. pyogenes for inducing double stranded breaks. For a more comprehensive list of our bacterial...see here . pGRG Series Various Plasmids Tn7 Transposition Nancy Craig A gene of interest cloned into one... one of these plasmids can be transposed into the attTn7 attachment site found in many enterobacterial...enterobacterial genomes. The bacteria of interest are transformed with the appropriate construct, grown at the permissive...

...Tools Atlas (Link opens in a new window) . Figure 1: Selection, prep, and testing of AAV enhancers in ...Omstead V, Oyama A, Pham T, Pom CA, Potekhina L, Ransford S, Rette D, Rimorin C, Rocha D, Ruiz A, Sanchez...Ting J, Tasic B. A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell...