Root > Products > StarGate Expression Vectors > "Two-step-cloning" via pENTRY > Donor Vector Generation > Technical information about sequencing primers for entry vectors

Type IIS Restriction Enzymes

Type II enzymes are one of the 4 (I-IV) types of recognized endonucleases, which cut DNA at a particular recognition site. Type II enzymes cleave within or a short distance from their recognition sites, which comprise usually 4–8 nucleotides in length.

Among them, are the type IIS enzymes, like LguI and Esp3I.

 

 

 

Type IIS restriction enzymes are dimeric enzymes that cleave DNA at a defined distance from their non-palindromic, asymmetric recognition site. This means that the target sequence can only be read in one direction. Thereby the digestion with only one single enzyme can generate two different independent sticky ends with 5’-overhangs allowing directional cloning. In addition, after digestion reaction the recognition sequence is removed completely and therefore the encoded amino acid sequence is not affected by remaining restriction enzyme sites.

The usage of type IIS restriction enzymes provides important features for cloning:

  • It allows one tube cloning
  • Expression of authentic proteins is possible (no additional amino acids)
  • The cloning will be always in frame with the vector features
  • Assembly of multiple fragments is possible

 

References

  1. Pingoud A, Jeltsch A (2001).  Structure and function of type II restriction endonucleases. Nucleic Acids Res. 29 (18): 3705–27.

Available Sequencing primers for Entry Vectors (Donor Vectors)

description amount cat. no.
Forward sequencing primer for pENTRY-IBA vectors; HPLC-purified 1 nmol 5-0000-153
Reverse sequencing primer for pENTRY-IBA vectors; HPLC-purified 1 nmol 5-0000-152
Forw. and rev. seq. primers for pENTRY-IBA vectors; HPLC-purified 1 nmol
each
5-0000-154