Part:BBa_K590010

pGA1A3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 1A3 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 1A3 vector.

Usage and Biology

This is a high copy plasmid backbone that confers ampicillin resistance. It was deposited in the registry with an insert coding for LacI-repressible GFP. This plasmid has been altered to conform to [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] standards, with arbitrary, Gibson-friendly sequences placed in between the restriction enzyme sites EcoRI-BglII, and BamHI-PstI.

Characterization

This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. We determined the cloning efficiency 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.

To understand how the copy number varies from plasmid to plasmid. The pGA vectors were transformed and the fluorescence levels were measured by flow cytometer. It was found that the fluorescence level is higher is high copy plasmid:

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2136
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2136
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2136
  Illegal BglII site found at 2151
  Illegal BamHI site found at 1
  Illegal XhoI site found at 16
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2136
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2136
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 1175