Part:BBa_K2549025

ZF43.8-VP64

This part is one of the downstream elements of our amplifier. Zinc finger 43.8 (Part:BBa_K2549048) is a synthetic engineered DNA binding domain with high sequence specificity and high orthogonality with other zinc finger proteins. VP64 (Part:BBa_K2549057) is a tetrameric VP16 transcription activator which shows ultrahigh transcription activation function. A SV40NLS domain (Part:BBa_K2549054) is placed on the N terminal of VP64 to guide the fusion protein to enter in the nucleus. A G4S linker (Part:BBa_K2549053) is set between ZF21.16 and SV40NLS. This part can also be utilized by other iGEM teams as zinc finger-based transcription activators to construct their own genetic circuits.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 304
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 187
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

It works as we designed

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Previous reported engineered zinc finger-based transcription factors

Lohmueller JJ et al have reported a tunable zinc finger-based transcription framework in mammalian cells.

Zinc finger-based transcription activator. Lohmueller JJ et al stated: We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression.

Zinc finger-based transcription repressor. Lohmueller JJ et al stated: We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression.

Artificial zinc fingers with modular DNA-binding domains

Collins JJ et al have reported a synthetic biology framework based on orthogonal artificial zinc fingers.

Collins JJ et al stated: Eukaryotic transcription factors (TFs) perform a variety of molecular functions to control promoters and facilitate the operation of genetic networks (top panel). Zinc fingers (ZFs) are modular domains found in many eukaryotic TFs that make sequence-specific contacts with DNA. Artificial ZF arrays were used as core building blocks for constructing synthetic TFs (sTFs) and gene circuitry in S. cerevisiae (bottom panel). The use of artificial ZF domains permits a fully decomposed design of a sTF, for which the molecular component properties are accessible, modular, and tunable (red italicized). The independent control of these component properties enables the systematic construction and modulation of transcriptional behavior. AD, transcriptional activation domain; GOI, gene of interest; REs, regulatory elements.

Collins JJ et al stated: sTFs constructed from OPEN-engineered ZFs are orthogonal to one another. sTF43-8 activated noncognate Promoter21-16 due to the fortuitous creation of a sequence that is significantly similar to the binding sequence of 43-8, when the downstream BamHI restriction site is considered.