...Addgene Resources on Addgene.org Find Yeast Expression Vectors Check Out Our Molecular Biology Reference...epitope tags to the C-termini of Met proteins, which can be used to confirm protein expression by western blotting...yeast species into the pYES2.1/V5-His yeast overexpression vector. This plasmid contains origins of replication... or S. cerevisiae. Students then determine if expression of the plasmid-encoded MET gene allows the transformed...cannot grow without uracil supplementation (6). Expression of MET/Met genes from the pYES2.1 plasmid is ...Students then use replica plating to determine if expression of MET/Met genes from the plasmids allows the...Since the GAL1 promoter controls MET/Met gene expression, complementation should only occur when galactose...

...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using Fluorescence-...BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP conversion and to target specific...integrated it into human and mouse cells that expressed either Cas9 or an optimized cytidine base editor... of a mutated start codon to initiate protein expression, it can be used for an array of detectable proteins...successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome would restore eGFP fluorescence, making...

...conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger expression cassettes ...chromosomal integration and gene expression, the system includes a toolkit to express recombinant proteins within...the Rapid Design and Production of Diverse Expression Vectors.” ACS Synthetic Biology, vol. 6, no. 7, American...journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library for Advanced... tools can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even...from individual transcription units (TUs) used to express your favorite gene-of-interest, all the way to ...with E. coli and/or cell-free systems. Protein expression in yeast MoClo is also for eukaryotes! The technology...

...targeted repression of gene expression. They successfully knock down expression of essential and non essential... of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen...) technology (Ronda et al., 2016). pMA7CR_2.0 expresses lambda Red and Cas9, which are separately inducible...function in bacteria, most efforts to regulate gene expression were limited to inducible promoters or direct... much more user-friendly way to modulate gene expression. The Marraffini lab and Qi lab developed early...repression. Using a Tet responsive promoter to drive expression of a catalytically dead Cas9 enzyme allows reversible...the gRNA cassette, one can reliably knockdown expression of multiple target transcripts simultaneously...

...13779 pRho-Cre Cre-Myc rhodopsin Mammalian Cepko 13780 pNrl-Cre Cre-Myc, Expressed in rod photorecetor...present. By expressing Cre at specific times or locations, you can precisely control expression of your gene...developed ways to tightly control Cre expression and to optimize Cre expression once it is induced. Addgene has...defines the areas in which Cre will be expressed. Cre may be expressed globally under a broadly active promoter... or expressed only in a subset of cells under a more specific promoter (e.g. Rho-Cre is expressed in the...visualization of Cre expression. Optimized Cre: Codon-optimized Cre ( iCre ) is expressed at higher levels...Cre expression when cloning in E. coli . This alteration enables the generation of a single vector containing...

...loxP sites such that their expression is turned on or off in cells expressing Cre. The result of a system...allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell...immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes...efforts have been made to create transgenic mice expressing fluorescent protein reporter constructs in the...above depicts the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after...Subcellular Localization Find Fluorescent Protein Empty Vectors ...system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals an essential role...

...continues to be challenging. Organism specific viral vector systems would also be beneficial for many systems...snail can be manipulated by injected RNA and can express fluorescent proteins. This lab has created an automated...habitats - even 50 meters from Old Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs...So, think beyond Drosophila melanogaster or Saccharomyces cerevisiae (both fine research organisms) -  ...

...Tetracycline (Tet) Inducible Expression Tetracycline (Tet) Inducible Expression Additional Resources: Browse...subsequent increase in expression by the activation domain, resulting in reduced gene expression. This idea of ... rtTA or TetR expression plasmid Stable cell lines can be made that continuously express a system component...Wiederschain 41393 pCW57.1 Lentiviral vector for inducible expression for Gateway cloning; selection cassette...Root 11651 pLVUT-tTR-KRAB Lentiviral vector for inducible expression of transgene or shRNA; see article ...Vogelstein 25735 pSLIK-Neo Lentiviral vector for inducible expression of a miR-shRNA; selection cassette ...On Fraser 44012 pInducer20 Lentiviral vector for shRNA expression; see article for additional tookit plasmids...

...: 95 °C for 15 min, 35 cycles of (95 °C for 30 sec, 60 °C for 1 min, 72 °C for 1 min), and 72 °C for 10...following parameters: 37 °C for 30 min; 95 °C for 5 min and then ramp down to 25 °C at 5 °C/min. Dilute oligos...parameters: Cycles 1-20 (37 °C for 5 min, 20 °C for 5 min); Cycle 21 (80 °C for 20 min). These cycling ...Incubate at 30 - 37 °C for 24 - 72 hr. 30 °C may enhance genome editing efficiency, but 37 °C is acceptable....the 20-mer is not G. sgRNA expression from the U6 promoter of the pX330 vector is enhanced by the inclusion...cells to incubate at 37 °C for 3 - 7 days and allow the clones to incubate at 37 °C for 7 - 14 days. Vary...thermocycler and run the following program: 65 °C for 6 min, 98 °C for 2 min to extract gDNA. Measure the DNA...

...pmScarlet_C1 - Mammalian Expression pCS2+mScarlet-C Cloning Vector - Mammalian Expression mScarlet-I 569 593...pBad-EBFP2 - Bacterial Expression EBFP2-N1 - Mammalian Expression EBFP2-C1 - Mammalian Expression EBFP2-pBAD - ...- Mammalian Expression pCyPet-C1 - Mammalian Expression pCyPet-His - Bacterial Expression CYPet-N1 - Mammalian...pEiLOV-N1 - Mammalian Expression pGEX iLOV - Bacterial Expression iLOV-N1 - Mammalian Expression iLOV-C1 - Mammalian...Mammalian Expression pCAG-GFP - Mammalian Expression pLV-eGFP - Mammalian Lentiviral Expression Ac5-STABLE1...1 - Mammalian Expression mEGFP-N1 - Mammalian Expression mEGFP-C1 - Mammalian Expression mEGFP-pBAD - ... - Mammalian Expression (this is a bicistronic vector rather than a fusion protein vector) ZsGreen1-N1...

... with viral vector manufacturing facilities, such as the University of Pennsylvania Vector Core (Link ...produced the viral vector lot. Contact us to determine which facility produced your viral vector lot. At Addgene...Transducibility Some viral vectors are tested in vitro and in vivo for gene expression and/or function. These...Empty vector particles can be identified after negative staining and appear darker than full vector particles...Electron micrograph of AAV vector preparation shows that the vast majority of the vectors consist of full particles...-80 °C. Titer Titering is either performed by Addgene or by the University of Pennsylvania Vector Core...80 °C. Titer All titering is performed on lentiviral preparations that have been stored at -80 °C and ...

... Empty Vectors Many gRNA empty vectors have been deposited at Addgene. To find the gRNA vector for your...constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 ...size and allow for expression of more sgRNAs in a single vector. sgRNAs are expressed as polycistronic ...gRNA expression vectors for insertion of custom gRNA target sequences. CRISPR...CRISPR Plasmids: Empty gRNA Expression Vectors Browse CRISPR Plasmids By Function Genome Editing Cut Base...by the Doudna Lab . Plasmid Expression System Cloning Enzyme Co-expressed Cas9 Cas9 System Selection PI...BbsI none S. pyogenes Gersbach pUC57-sgRNA expression vector 51132 Mammalian BsaI none S. pyogenes Huang...

...j.bbrc.2013.08.013. Empty gRNA Expression Vectors Select a gRNA expression plasmid based on factors such...choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the...using does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the...Libraries gRNAs Empty gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish...repressor peptide like KRAB, can knock down gene expression by interfering with transcription. Design your...cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in...both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single...

...cloning vector Retroviral vectors: neomycin (pSIR-neo) , GFP (pSIR-GFP) , DsRed (pSIR-DsRed-Express2) , human... sgRNA dual expression: pAC2 , pAC152 , pAC153 , pAC154 ; pmax dCas9 activator expression: pAC91 , pAC92... pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9-Blast and lentiGuide-Puro...protocol Church gRNA design and cloning gRNA cloning vector PDF 115.2 KB Church gRNA design and cloning for...human pluripotent stem cells pCas9_GFP ; gRNA empty vector Link O'Connor-Giles Fly: gRNA and ssODNs design...pX330) Link Parrott NEBuilder Assembly of CRISPR vectors using ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H...design and cloning All-in-one plasmid containing expression cassette for NmCas9 and BsmBI site for cloning...

...Marker PI Publication Empty gRNA Expression Vectors Select a gRNA expression plasmid based on factors such...choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the...using does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the...Libraries gRNAs Empty gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish...CRISPR expression plasmids that have been designed for use in plants. CRISPR...repressor peptide like KRAB, can knock down gene expression by interfering with transcription. Design your...cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in...

...Publication Empty Prime Editing gRNA Vectors A selection of empty gRNA vectors suitable for prime editing are...below. Use the search bar to find a gRNA vector based on expression system, promoter, the type of gRNA (e.g...Cas9. ID Plasmid Expression System Promoter Guide RNA Type Cloning Enzyme Co-expressed Cas9 Selection PI...Libraries gRNAs Empty gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish... editing plasmids. Plasmids are available for expression in mammalian systems, bacteria, plants, and Drosophila...which a reverse transcriptase (RT) is fused to the C terminus of Cas9 H840A nickase. The fusion enzyme ...-Puro Mammalian, piggyBac hU6 pegRNA BsmBI No Puromycin Jacob Giehm Mikkelsen 173222 pPBT-PE2-PuroTK-pegRNA_GG...

... T H A, C, or T K G or T M A or C N A, T, C, or G R A or G S C or G V A, C, or G W A or T Y C or T Amino...Commonly used cloning vectors include Gateway entry vectors and TOPO cloning vectors. If you are looking...information. Expression Plasmids - Used for gene expression (for the purposes of gene study). Expression vectors...amount of protein or RNA produced. Expression vectors can drive expression in various cell types (mammalian... the level of insert expression (i.e., a strong promoter directs high expression, whereas weaker promoters...for the transcription process to stop. An expression vector can also include an enhancer sequence which...reducing the expression of an endogenous gene. This is frequently accomplished through expression of an shRNA...

...reporter vector expressing firefly luciferase. pCMV-FLuc - Retroviral reporter vector expressing firefly...Lentiviral vector expressing destabilized EGFP. Lenti-luciferase-P2A-Neo - Lentiviral reporter vector expressing...IRES-ZsGreen-W - Lentiviral dual reporter vector expressing firefly luciferase and ZsGreen. NL4-3 mCherry...mCherry Luciferase - Lentiviral dual reporter vector expressing mCherry and firefly luciferase. pLentiEGFPdestablized...Viral Vectors 101: Pseudotyping Antibody Neutralization Response Against Pseudoviruses Expressing SARS-...can then be used to infect permissive cells that express ACE2, the cellular receptor for SARS-CoV-2, in ...Resources page for more information. Plasmids Expressing Spike and Spike Variants Full-length wildtype...

...Promoter PI Publication Empty gRNA Expression Vectors Select a gRNA expression plasmid based on factors such...choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the...Libraries gRNAs Empty gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish...cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in...both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single...On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety...Promoter Cloning Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes...

...codon-optimized for expression in bacteria and cloned into an IPTG-inducible His-tag expression vector. All His-tags...sustainable MDH-related CURE focusing on protein expression, structure and function, kinetics and mechanisms...His-tags are cloned at the C-terminus of MDH and, if indicated on the plasmid map, a TEV cleavage site ...Organism or species (e.g., human, watermelon, Streptomyces ) Subcellular compartment (chloroplastic, cytoplasmic...