Vectors multiple cloning site

Fig. 1. The expression vector pTDI. (a) pTDI vector map. (b) DMA sequence of the pTDI vector around the multiple cloning sites.

Expression vector with suitable promotor, multiple cloning site, and fusion tag, where applicable (e.g., six-histidine tag). 

Targeting vector A vector containing a positive selection marker (e.g., neomycin-resistance gene), negative selection marker (thymidine kinase gene) and a multiple cloning site. 

Over several decades, multiple vector systems for recombinant gene expression in E. coli have been developed. Modem vectors suitable for recombinant protein production vary in the used promoter system in the presence or absence of coding sequences for affinity tags upstream or downstream of the multiple cloning site (MCS) and of sequences coding for leader peptides for the protein export. Moreover, different origins of replication (ori), antibiotic selection marker genes and MCS are used. 

Fig. 4 Plasmids for the intracellular production of tagged proteins in B. megaterium. All plasmids of the series 1622 are based on the shuttle vector pSTOP1522 (Fig. 3). (a) DNA sequence of ribosome binding site (RBS) and multiple cloning site (MCS) of the expression plasmid pSTOP1622. The...

In this experiment, you will amplify a fragment of pBluescript II (a plasmid), which includes the multiple cloning site (MCS) of the vector (Fig. 24-2). The pBluescript II plasmid comes in the S/K form and the K/S form. These two plasmids are identical except for the orientation of the MCS (see Fig. 24-2). Using restriction enzymes and agarose-gel electrophoresis, you will determine which of these two plasmids was used as a template in the PCR reaction. The sequences of the two primers that will be used in the PCR reaction are shown under Supplies and Reagents. Primer 1 will anneal to positions 188 to 211 (5 to 3 ) on one strand of the plasmid, while Primer 2 will anneal to positions 1730 to 1707 (5 to 3 ) on the opposite sttand of the plasmid (Fig. 24-3). On amplification, a 1543-base-pair fragment of DNA will be produced that includes the multiple cloning site of the plasmid. fl (an isoschizomer of SacT) and Kpnl will then be used to determine whether the S/K or K/S form of the pBluescript II plasmid was used as a template in the amplification reaction. 

Fig. 24.6 Expression vectors and the generation of fusion proteins. Expression vectors have optimized all the signals required for transcription (inducible promoter and transcriptional terminator) and for translation (ribosome binding site). Some of them carry the gene for (5-galactosidase with a multiple cloning site that allows the insertion of small genes for the generation of fusion proteins.

The pUC vectors contain an ampicillin-resistance gene and a multiple cloning site at the 5 -end of the lacZ gene. Insertional inactivation of lacZ allows... 

Figure 2 A general overexpresion vector. Most Escherichia coii vectors for recombinant protein production will have most of these features. The N- and C-terminal fusions and associated protease cleavage sites are optional. The multiple cloning site (MCS) may be replaced by DNA sequences for site-specific recombination in some vectors.

Figure 6 Different multiple cloning sites in different vectors, (a) MCS from pET-28a (Novagen, Madison, Wl). (b) MCS from pGEX-4T-1 (GE Healthcare, Piscataway, NJ). (c) pASK-IBA6 (IBA, Germany).

The sequencing results of the fragments inserted into PUCD615 vector also revealed that the uvrA, alkA genes had been inserted into the multiple clone sites correctly, the insert direction and the reading frame were also correct. So the recombinant PUCD- vrT and PUCD-a/fcf vectors were constructed successfully. 

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