Part:BBa_K590013

pGA4A5, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 4A5 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].

Usage and Biology

This is a low copy plasmid backbone which has Ampicillin resistance, with pLac promoter and 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

To understand how the copy number varies from plasmid to plasmid, the pGA vectors were transformed into a lacI knockout (strain [http://cgsc.biology.yale.edu/Strain.php?ID=16959 2.320]) and the fluorescence levels were measured by flow cytometry. As expected, we found the low copy pGA4A5 plasmid to express GFP at levels lower than medium and high copy plasmids.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3371
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3371
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3371
  Illegal BglII site found at 3386
  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 3371
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3371
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 3023