An MCS is a short DNA sequence consisting of restriction sites for many different restriction endonucleases. MCS escalates the number of potential cloning strategies available by extending the range of enzymes that can be used to generate a restriction fragment suitable for cloning. By combining them within a MCS, the sites are made contiguous, so that any two sites within it can be cleaved simultaneously without excising vector sequences. The MCS is inserted into the lacZ sequence, which encodes the promoter and the α-peptide of β-galactosidase. Insertion of the MCS into the lacZ fragment does not affect the ability of the α-peptide to mediate complementation, while cloning DNA fragments into the MCS does. Therefore, recombinants can be detected by blue/white screening on growth medium containing X gal in presence of IPTG as an inducer.

Fig 1-3.7.1.B : pUC plasmid

1-3.8 Expression Vector:

A vector used for expression of a cloned DNA fragment in a host cell is called as an expression vector. These vectors are frequently engineered to contain regulatory sequences that act as promoter and/or enhancer regions and lead to efficient transcription of the insert gene. Commonly used expression vector series are: pET vectors, pBAD vetors etc.

Fig 1-3.8 : Expression vector

For an expression vector following features are essential:

• Promoter: Promoter is a sequence which is recognized by sigma subunit of RNA polymerase which is required for initiation of transcription of gene of interest.
  
  
• Terminator: It is a DNA element present at the end of a gene where transcription of gene ends. Terminator is short nucleotide sequences which can base pair with itself to form hair pin loop.
  
  
• Ribosome binding site: It is a short nucleotide sequence recognized by the ribosome as the point at which it should attach to the messenger molecule. The initiation codon of the gene is always a few nucleotides downstream of this.