Part:BBa_K731700

Platform for terminators analysis under the control of T7 promoter

We used this part to test terminators: the two subsequent proteins allow termination efficiency's quantification as the ratio between Venus's and Cherry's fluorescence. 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, and mutated the two illegal sites present in the original plasmid.

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_K731710, where the T7 promoter has been replaced with 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 6850
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 6856
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6850
  Illegal BglII site found at 6011
  Illegal BamHI site found at 6832
  Illegal XhoI site found at 753
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 6850
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 6850
  Plasmid lacks a suffix.
  Illegal XbaI site found at 6865
  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 6800
  
• 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