Part:BBa_K2205026

pSB1A51 - Promoter Library Screening Plasmid

Screening plasmid for the construction and screening of a biobrick compatible promoter library. This part is an improvement of a previous promoter library screening plasmid (J61002). The improvements made are as follows:

1 – A bi-directional terminator has been inserted upstream of the iGEM cloning site to prevent any leaky expression from the plasmid backbone and to prevent expression from the promoter in the reverse direction.

2 – The Lac Operator sequence has been inserted immediately downstream of the promoter insertion site, this is to allow for the expression of the promoter to be repressed until the user desires to test the construct.

3 – The ribosome binding site (B0034) is inserted between two restriction sites (SacI and BamHI). This is to allow for the RBS to be changed at will if the user wishes to screen a library of RBS as well as promoters.

4 – The reporter gene used is sfGFP, this is a shorter gene sequence which produces a brighter fluorescence than mRFP. The emission wavelength of sfGFP is (509 nm) which is further from the standard cell density wavelength (600 nm) than the emission wavelength of mRFP (584 nm), this makes interference from high cell density samples less likely.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 96
  Illegal XbaI site found at 111
  Illegal SpeI site found at 119
  Illegal PstI site found at 979
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 96
  Illegal SpeI site found at 119
  Illegal PstI site found at 979
  Illegal NotI site found at 102
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 96
  Illegal BamHI site found at 173
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 96
  Illegal XbaI site found at 111
  Illegal SpeI site found at 119
  Illegal PstI site found at 979
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 96
  Illegal XbaI site found at 111
  Illegal SpeI site found at 119
  Illegal PstI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2060
  Illegal SapI.rc site found at 191