Part:BBa_K731700:Design

Platform for terminators analysis under the control of T7 promoter

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
  

Design Notes

When using and modifying this construct it is important to remember that the two proteins have strong C-terminal homology. This should be taken into consideration when designing primers. The two proteins have partially overlapping emission and absorption spectra and different folding kinetics.

	Excitation (nm)	Emission(nm)
mCherry	587	610
Venus	515	528

Source

The part was built starting from a construct kindly provided from the Mansy lab made by Roberta Lentini(RL024). The construct is a pET21b where mCherry and A206K Venus were inserted with a 20 bp spacer between the 2 proteins. That plasmid was mutated two times to eliminate illegal restriction sites and a prefix-suffix linker was added by mutagenesis.