Part:BBa_K731710

Platform for terminators analysis under the control of tac promoter

This part is used to analyse terminators: two subsequent fluorescent proteins (mCherry and A206K Venus)allow to obtain several information about terminator's presence effect.

The vector is pET21b, that contains lacI, t7 promoter and ampicillin resistance gene.We inserted the prefix-suffix linker between the two proteins, to allow terminator's characterization, mutated the two illegal sites present in the original plasmid and replaced T7promoter with Ptac.

The two fluorescent proteins have partially overlapping emission spectra, thus, to avoid unwanted interferences, the fluorimetric measures should be taken with these parameters: Venus excitation: 485nm, Venus emission: 528nm Cherry excitation: 528nm, Cherry emission: 615nm.

This is the twin construct of BBa_K731700, that has T7 promoter instead of Ptac. The two constructs allow terminators' characterization with different RNApolymerase and analysis of any potential difference. We tested both constructs in the E.Coli strain BL21pLysS, that has both Coli and T7 polymerases.

Sequence and Features

Assembly Compatibility:

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

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6865
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 6871
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6865
  Illegal BglII site found at 6011
  Illegal BamHI site found at 6847
  Illegal XhoI site found at 753
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 6865
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 6865
  Plasmid lacks a suffix.
  Illegal XbaI site found at 6880
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 714
  Illegal NgoMIV site found at 1051
  Illegal NgoMIV site found at 4231
  Illegal NgoMIV site found at 4391
  Illegal NgoMIV site found at 5979
  Illegal AgeI site found at 6815
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 2228
  Illegal SapI.rc site found at 3310