Part:BBa_K4235002

pFastBac-Htb_miniTn7

Usage and Biology

The plasmid pFastBac is used for the expression of 6x His-TEV-tagged proteins in insect cells through the Bac-to-Bac baculovirus expression system. The main components of this vector are an Ampicillin resistance marker gene and a mini-attTn7 transposon segment, which contains the multiple cloning site for expressing recombinant proteins driven by the polyhedrin promoter, a SV40 polyA signal and Gentamicin resistance gene expressed from the pC promoter. The mini-attTn7 segment is flanked by terminal inverted repeats called Tn7-L and Tn7-R which are recognized by the enzyme transposase. Transposases are responsible for introducing double-stranded breaks at these mini Tn7 elements and freeing the DNA sequence from the transfer vector. This DNA segment can then be inserted/transposed into another bacterial or baculovirus genome propagated in E coli DH10Bac cells, allowing for the production of recombinant Bacmid particles.

This plasmid was modified by the Airola lab at Stony Brook University to have twin strep tags and a 6x His-tag with a TEV site on the C-terminal of the MCS instead of N-terminal, as found in the original pFastBac HT-B.

For our project, we intended on cloning our insert BBa_K4235000 just upstream of the twin strep tag and 6x His-tags which are on the C-terminal of the MCS in YmBac-II.

This vector contains the following parts:

• Polyhedrin Promoter: BBa_K4235001
  
• Gentamicin resistance cassette (pC+GmR): BBa_K4235024
  
• Ampicillin resistance cassette (AmpR promoter+CDS): BBa_K4235025
  
• SV40 polyA signal: BBa_K4235020
• miniatt-Tn7 transposon segment (polyhedrin+MCS+SV40polyA+GmR cassette): BBa_K4235010

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4069
  Illegal XbaI site found at 4116
  Illegal SpeI site found at 4097
  Illegal PstI site found at 4124
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4069
  Illegal SpeI site found at 4097
  Illegal PstI site found at 4124
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4069
  Illegal BglII site found at 2547
  Illegal BglII site found at 3017
  Illegal BamHI site found at 4062
  Illegal XhoI site found at 4131
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4069
  Illegal XbaI site found at 4116
  Illegal SpeI site found at 4097
  Illegal PstI site found at 4124
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4069
  Illegal XbaI site found at 4116
  Illegal SpeI site found at 4097
  Illegal PstI site found at 4124
  Illegal NgoMIV site found at 127
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1304
  Illegal BsaI site found at 3661
  Illegal SapI.rc site found at 2386
  

Source

We received this plasmid as a generous donation from the Airola Lab at Stony Brook University.