Part:BBa_K4364000

mRFP1-based cassette for TA cloning with dual T7 promotors

This part is a modified version of part BBa_K3764000

This part can be used to create a T-vector with two T7 promotors that is compatible with various TA cloning protocols. In order to do this, it should be cloned in a suitable pSB vector, this plasmid must be extracted and then treated with the restriction endonuclease AhdI. It will produce a linearized vector with single T overhangs. Moreover, the TA-cassette is integrated into the reading frame of mRFP1 under the control of the lac promoter. The empty vector produces notable red-colored colonies. If the TA cloning is successful the positive transformants will be white.

Please be careful if you transfer this part to pSB_A_ plasmid backbones - the beta-lactamase encoded by these vectors contains an AhdI site that should be destroyed first.

Purpose

The major purpose of this part is to be used in combination with the MutaT7 bacterial strain (Addgene #156460). It is designed to allow efficient random mutagenesis of the cloned sequence to improve the properties of the metallo-beta-lactamases that participate in the CADABRA enzyme cocktail for carbapenem removal.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 199
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 207
  Illegal BamHI site found at 316
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 906
  Illegal AgeI site found at 1018
  
• 1000
  COMPATIBLE WITH RFC[1000]