Part:BBa_K1890020

GFP with strong constitutive promoter, RBS and terminator

Indroduction

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria. This mutant represents a series of mutations resulting in enhanced maturation and emission [1]. It is expressed under control of the strong constitutive promoter BBa_J23100, strong RBS BBa_B0030 and terminators BBa_B0010 and BBa_B0012.

Construction

This part is based on the part BBa_E0840, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter BBa_J23100. Two PCR reactions were performed with the following primers (Table 1).

Table 1: Primers used to add promoter to GFP BioBrick.

Primer name	Sequence
E0840_FW	ATTAAAGAGGAGAAATACTAGATGCGTAAAGG
J23100-E0840_FW	CGGCGAATTCGCGGCCGCTTCTAGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCATTAAAGAGGAGAAATACTAGATGCGTAAAGG
VR	ATTACCGCCTTTGAGTGAGC

To prevent annealing of the primer containing the promoter (J23100-E0840_FW), to the BioBrick prefix, a preliminary PCR was performed with a forward primer not containing the BioBrick prefix (E0840_FW) (Figure 1).

Figure 1: Construction of the biobrick K1890020 by means of two PCRs, using biobrick E0840 as a template.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 700
  

References

[1] Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.