Part:BBa_K2549056

destroyable nuclear location sequence

This part is a destroyable nuclear location sequence. It derives from a bipartite nuclear targeting sequence which has identified two basic interdependent domains separated by 10 amino acids spacers which tolerate point mutations and some insertions. We replaced the spacer with a high-affinity TEV cleavage sequence to make it destroyable. Besides, we insert glycine and serine on both ends of the TEV cleavage sequence to ensure higher flexibility. When coexpressed with TEV protease in the same cell, the destroyable nuclear location sequence is cleaved, thus destroying the fusion protein and making it unable to enter in the nucleus.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

Both Part:BBa_K2549039 and Part:BBa_K2549040 have this peptide sequence.

TEVp-based IMPLY gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .

We show that when KRAB-dNLS-ZF21.16 (Part:BBa_K2549040) exist while TEVp (Part:BBa_K2549041) did not, the expression of d2EGFP was repressed by KRAB. When TEVp and KRAB-dNLS-ZF21.16 both were present, TEVp cleaves dNLS and prevents KRAB functional, although the release from the suppression was not as strong as we hoped. We need to improve this part.