Part:BBa_K2984052

L1c-PsadIntron-bleRscp-ARS-YFP-Rbcs2

This vector is a part of the Chlamy-HUB-Collection. Level 1 construct containing secretion signal of arylsulfatase ARS fused to the flourescent protein mVenus. To influence the expression of mVenus, a bleomycin resistance gene is connected through a self cleaving peptide (scp) to the part. The Promoter is the PsaDintron. The construct ends with the Rbcs2 terminator.

The bleomycin resistance leads to a higher expression of the enzyme, due to its working mechanism. For every two bleomycin molecules, the bleomycin resistance gene must be expressed once. The resistance works in a ratio of 1:2 to the antibiotic (Hayes et al., 1990). By having the resistance in the same open reading frame, the enzyme is also expressed, leading to a higher expression of the enzyme (Lumbreras et al. 1998).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2051
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2051
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2051
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2051
  Illegal NgoMIV site found at 1607
  Illegal NgoMIV site found at 2528
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Fig.1 - Image of a successful transformation in E. coli after ligation of the construct. The construct can then be isolated from E. coli to be transformed in C. reinhardtii.

References

1. Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.
2. Hayes, J. D., & Wolf, C. R. (1990). Molecular mechanisms of drug resistance. Biochemical Journal, 272(2), 281.