Vector destination

Type of expression vector Destination of expressed protein Regulation of protein biosynthesis Piasmids, mitochondria, genome Intraceiluiar, perisplasm, exracellular Constitutive expression, induction... 

The reaction differs from excision of the X chromosome because the Entry Clone contains two attL sites and the destination vector contains two attR sites (Hartley et al., 2000). The att sites are mutated to ensure recombination only occurs between attLl and attRl and between attL2 and att.R2. The recombination reaction proceeds through a cointegrate molecule that is resolved to create a destination vector containing the gene of interest with the desired promoter and tag sequences (Fig. 4.6). 

Figure 4.6. Recombinational cloning (RC). A. Cloning of PCR products using the attB + attP > attL + attR reaction catalyzed by Int and IHF. The result is an Entry Clone that can be used to create functional vectors. B. Conversion of an Entry Clone to a functional vector using the attL + attR > attB + attP reaction catalyzed by Int, Xis and IHF. A wide variety of functional vectors can be constructed by using a Destination Vector with the appropriate promoters and tags.

Destination vector. A vector for expression of a cloned ORF in an organism of interest. This vector should have the ccdP> cassette at the position where the ORF is to be inserted, as is the case for a Donor vector see Note 3). 

Many destination vectors are commercially available. However, it is also possible to construct a destination vector that is suitable for individual experiments. To constrnct snch vectors, one should insert the rr ffi-containg cassette (commercially available from Invitrogen) into the appropriate locns of a Gateway-incompatible vector. The resultant plasmids can be amplified only in the DB3.I strain, due to the toxic ccd gene (7). 

The concentration of an Entry clone plasmid should be kept at a low level (1-10 ng/pi) in the LR reaction. A high amount of an Entry clone proved to be adverse, contrary to our expectations. Similarly, the concentration of the destination vector should be kept low. In contrast to the LR reaction, it would be better to use the insert DNA (PCR products) at a high concentration in the BP reaction. 

Vector DNA 150 ng/pL pGEX-6PDES A+destination vector Store at -20°C. 

The method by which a DNA vector is trafficked intracellularly may have important implications on the efficiency of gene delivery. If DNA vectors are trafficked to sites far from the nucleus, gene delivery will be hindered by the diffusion-limited cytoplasm. PEI and polylysine are both cationic polymers with the ability to condense DNA however, they have been shown to traffic to different intracellular destinations [198] and result in different transfection efficiencies [199]. In addition, particles of different chemistries were routed differently by the same cell type [200]. Understanding the intracytoplasmic transport of DNA vectors will be critical if higher-efficiency vectors are to be engineered. 

To transfer the DNA fragment from an entry vector to another plasmid, the entry vector is mixed with a destination vector (that carries attRl and attR2 sites), after... 

DNA from the resulting colonies is then screened for the correct cassette orientation (the ligation is bi-directional) by restriction enzymes or PCR. Once the properly oriented clone is isolated, a destination vector is born. 

For a complete list of commercially available destination vectors, please visit uniw.in-vitrogen.com. These cover a broad array of applications including protein expression in E. coli, yeast, insect, and mammalian cells, yeast two hybrid protein interaction, and gene knock-down RNAi vectors. 

Modular assembly of expression constructs including promoters, ORFs, epitope and purification tags, can now be achieved in HTP, without the use of restriction enzymes and ligase. Once a set of entry vectors has been created, the elements can be mixed and matched in an LR reaction. Elements in a multi-fragment constract can be selectively removed via a specific BP recombination reaction, thereby creating an intermediate destination vector. For example, an enzymatic pathway can be assembled using modular promoters and ORFs to create a synthetic operon. Once the wild-type activity is established, a library of mutants can easily replace specific... 

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