Part:BBa_K3582500

Modified Pet28a(+) expression vector

pET28a(+) is a novel expression plasmid generally used in E-coli strains for protein production purposes. It has a kanamycin resistance gene as a selection marker. Plasmid sequence consists of the T7 promoter and terminator sequences as well as lac operon sequence and lac promoter for protein expression induction. Apart from this, it also has well-defined operator, promoter, MCS and tag sequences precisely inbuilt.

The normal pet28a(+) vector contains several unnecessary components like 6Xhis tag, thrombin site etc. that could interfere with protein expression and purification. Identifying a proper insertion site becomes difficult. The normal version does not even satisfy the RFC 10 criteria.

This version of Pet28a(+) vector is stringently modified by removing all the unnecessary tags and non required regions. The insertion site is aptly annotated with a start and a stop codon where the insertion of the ORF can be made. All of the other features of the vector are conserved and undisturbed.

Figure 1. Visualiztion of modified Pet28a(+) vector with complete annotation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 105
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 137
  Illegal NgoMIV site found at 1725
  Illegal NgoMIV site found at 1885
  Illegal NgoMIV site found at 4932
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2805