...developed cloning vectors to harness it. These vectors, called pKIL18 and pKIL19, contained the ccdB gene in frame...non-recombinant vectors (re-ligated empty vectors, for instance) would still express ccdB and therefore ...toxic effects of CcdB. Another commercially available, CcdB-resistant strain is ccdB Survival™ from InvitrogenTM...DB3.1 and ccdB Survival strains have been developed specifically with ccdB-containing vectors in mind, ... the popular GatewayTM method possible: ccdB. But what is ccdB, what role does it play in modern cloning...interest. Simply stated, ccdB makes cloning easier by selecting against vectors that did not take up your...harnessed it to evolve cloning technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor ...

... of Gateway cloning vectors Vector type  Vector features Purpose Donor vector attP sites for recombination...sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The ccdB gene is present in the donor vectors and the destination...sequences into multiple vector systems. With the appropriate entry and destination vectors, one can use Gateway...byproduct of the reaction, the ccdB gene is excised from the donor vector.   Figure 2: The Gateway...DNA fragment containing the ccdB gene is excised from the destination vector.  Once the BP and/or LR reactions...BP or LR reactions. Since the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most ...donor vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO...

... of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is ...the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists of CcdB (toxin...cloning, where the ccdB gene serves as a counterselectable marker in the destination vector. Successful cloning...when the ccdB gene is replaced by the gene of interest. As a result, cells that retain the ccdB gene die...toxin) and CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA...a higher percentage of transformants contain the ccdB-less construct. Other toxin-antitoxin systems and...and their applications MazE/MazF Aside from CcdB/CcdA, there are many protein-based toxin-antitoxin systems...

... plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids...biosensor affinity for calcium.”  Primary Vector Type The vector type should describe how your plasmid ...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...should propagate your plasmids in: DH5α, NEB Stable, ccdB Survival, Pir1, or other. Whenever possible, we ...

...Genotype ccdB Survival 2 T1R   TOP10 derivative. For propagating plasmids expressing the ccdB gene (important...of protein synthesis Ptrc-ccdA   Propagation of ccdB-containing plasmids Hte   High transformation ...derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning). F- gyrA462 ... into and storage of lentiviral and retroviral vectors or cloning or repeated sequences with the potential...storage of unstable DNA and lentiviral/retroviral vectors. Used by Addgene for pooled library amplification...

...regions Joshua Mendell 212936 pGL3 Basic Vector Firefly Vector for investigating regions controlling transcription...interest are cloned into a Firefly luciferase reporter vector via Gateway cloning to test for the presence of...cloning. Lentiviral. Jorge Ferrer 87067 pcDNA3.1-ccdB-Nanoluc NanoLuc® Creation of N-terminal Nanoluc ...using Gateway cloning Mikko Taipale 87075 pLenti6.2-ccdB-Nanoluc NanoLuc® Creation of C-terminal Nanoluc ... Lentival. Mikko Taipale 87078 pLenti6.2-Nanoluc-ccdB NanoLuc® Creation of N-terminal Nanoluc fusions ... co-expressed. Oskar Laur 87070 pcDNA3.1-Nanoluc-ccdB NanoLuc® Creation of N-terminal Nanoluc fusions ...using Gateway cloning Mikko Taipale 87065 pcDNA3.1-ccdB-Renilla Renilla Creation of C-terminal Renilla luciferase...

...plasmids containing the ccdB gene, such as Gateway vectors, we recommend the ccdB Survival strain. Figure...the vector backbone you cloned your insert into and fill out the appropriate boxes with the vector backbone...backbone manufacturer, vector backbone size, total size of your vector + insert, any modifications you ...provide any full or partial plasmid sequences and any vector maps. The more sequence data available, the better... was a part of the insert (when cloned into the vector backbone). If your plasmid has more than one insert...you made to the backbone, and the vector type. Pro Tip! Reference the Addgene ID in the "Backbone Manufacturer...enter "Feng Zhang (Addgene plasmid #52961)".   The vector type should describe how your plasmid is intended...

...screening overview blog post.) *Note: Two Popular Vectors for Blue White Screening Available at Addgene Are... plasmid, creating an α-complementation cloning vector. When a cloning reaction goes to plan and your ...pUC18 and pUC19, and pBluescript are a few common vectors. Blue-white screening is just that – a screening...insert, there are other methods. Positive selection vectors encode a gene which, when expressed, is lethal ...plasmid containing the lethal gene. An example is the ccdB gene system commonly used in Gateway® Cloning. There...

...mostly due to the use of toxic ccdB to prevent any non-recombinant vectors from propagating. The ligation-independent...and colleagues. Here you simply PCR-amplify your vector and your insert, DpnI digest away the templates... time is of the essence. This method requires a vector backbone containing a topoisomerase I cut site,...the type IIS enzymes in the reaction to redigest vector reseals and other incorrect products. This feature...Addgene.org0 Check out protocols for plasmid cloning Find vector backbones Read our molecular biology reference ...

...starting vector that the depositing lab modified to create their new plasmid.  The Addgene Vector Database... modifying a vector previously deposited with Addgene, you can list the original vector’s plasmid number...Database has more information about common vector backbones.  The backbone modifications listed can refer ...transcribed and translated insert. If it is an empty vector with a tag, it’s terminus will be listed as the...section provides useful information about how the vector was assembled, and enables requesting labs to utilize...strain DH5a, but others may require strains such as ccdB survival (for Gateway plasmids).  We also like to...need extra stability, such as some of our viral vectors, in NEB Stable cells. Resource information: This...

...other molecules to host organisms. For instance, vectors derived from the Agrobacterium Ti virulence plasmid...developing new cloning techniques based on these vectors (Shareck et al., 2004). Plasmid categorization...coli fertility plasmid that also encodes a toxin, CcdB, and the resistance to that toxin, CcdA. Knowing..., Y., Lee, B., & Miguez, C. B. (2004). Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic ...

... Empty Vectors Many gRNA empty vectors have been deposited at Addgene. To find the gRNA vector for your...Multiplex gRNA Vectors Several systems have been developed to generate multiplex gRNA vectors. These systems...single gRNA vectors with various promoters, plasmids for assembling multiple gRNAs into one vector, and plasmids...expression vectors for insertion of custom gRNA target sequences. CRISPR... CRISPR Empty gRNA Vectors CRISPR Plasmids...Plasmids: Empty gRNA Expression Vectors Browse CRISPR Plasmids By Function Genome Editing Cut Base Edit...-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila ...

... cloning to form the desired vector. The pCoofy vectors contain the ccdB cassette, which is not copied... existing transposon and vector systems and creating an all-synthetic vector that included only the elements... into a destination vector in a predefined order. Finally, the destination vectors are used with Multisite... eight entry vectors can be used for each Multisite Gateway compatible destination vector for a maximum...step, compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional...of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers from Table...homology between the vector and gene primer for SLIC cloning. Amplify the pCoofy vector and the sequence ...

...compatible Y2H vector - BD pRPR1_gRNA_handle_RPR1t - gRNA expression empty vector pFA6a vectors - Epitope ...expression vector URA3 Yeast pAG306GAL-ccdB - Yeast Gateway expression vector (see more ...pFastBac Dual LIC - Vectors from the Gradia lab pFB-LIC-Bse - Insect expression vector from Gileadi lab ...expression vector with N- or C-terminal HA tag pInducer20 - Tet-inducible HA-tagged lentiviral vector for ORF...Gateway destination vector for bacterial expression; His,GST tag pE2c - Modified pENTR vector for C-terminal...-1-3xMyc - Bacterial vector for Myc tag pETcon(-) - Yeast surface display vector with a C-terminal Myc...His-tagged versions of pFastBac LIC vectors and pFastBac Dual LIC vectors for insect expression Fluorescent...

...destination vector (in this case the destination vector would be the lentiviral transfer vector). The destination...contains a Gateway cassette containing the ccdB gene, a ccdB survival strain is necessary. What cell line...insert of interest from its parent vector and ligating into a second vector in such as way that the insert...later be digested from this new vector and cloned into the lentiviral vector. This is basically shuffling...between vectors until the gene of interest is flanked by sites compatible with those in the vector into ...site (MCS) from a separate vector into the single site in the lentiviral vector and generate more useful...by the design of the vectors and by safe laboratory practice. In terms of vector design, 2nd and 3rd generation...

...Commonly used cloning vectors include Gateway entry vectors and TOPO cloning vectors. If you are looking...combination of a DNA fragment with a plasmid or vector DNA backbone generates a recombinant DNA molecule...specifically engineered plasmids, commonly referred to as vectors, which have become possibly the most ubiquitous...facilitate the cloning of DNA fragments. Cloning vectors tend to be very simple, often containing only a...expression (for the purposes of gene study). Expression vectors must contain a promoter sequence, a transcription...the transcription process to stop. An expression vector can also include an enhancer sequence which increases... amount of protein or RNA produced. Expression vectors can drive expression in various cell types (mammalian...