Part:BBa_K590014

pGA3K3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 3K3 plasmid backbone that was made by [http://2011.igem.org/Team:Washington UW iGEM Team 2011] as part of the [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly toolkit]. Using this plasmid increases the efficiency for anyone doing Gibson cloning into a 3K3 vector.

Usage and Biology

This is a medium copy plasmid backbone that confers kanamycin resistance. It was deposited in the registry with an insert coding for LacI-repressible GFP.

Below is the gel image of the plasmid amplified with universal pGA backbone primers pGAsuffix_fwd and pGAprefix_rev. The band is near the expected length of 2750 bp.

1kb Ladder (left), pGA3k3 (right)

Characterization

A sister plasmid (pGA1A3) with the same BglBrick prefix and suffix regions has been shown to have much higher efficiency than the equivalent pSB vector. In a pGA vector evaluation, we determined the cloning efficiency by by dividing the # of bright colonies by the (# of total colonies - # of background colonies). The background colonies were determined by a control sample containing 100 picograms of just the pGA backbone. See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal NheI site found at 1384
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal BglII site found at 2744
  Illegal BamHI site found at 1
  Illegal XhoI site found at 16
  Illegal XhoI site found at 177
  Illegal XhoI site found at 1020
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal AgeI site found at 1470
  Illegal AgeI site found at 1793
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2498