Part:BBa_K2066004

Tet Monomer C w/ 8 bp spacer for ICA

The part is flanked by UNS 2 and UNS 3 as per the WM UNS gibson cloning standard. This part is the TetO, 8BP spacer 'C' monomer for use in ICA to create repeated TetO Arrays of variable length. The monomer can be PCR amplified using UNS 4 and UNS 5 PCR landing pads. Next, the part should cut with BsmBI restriction enzyme to expose sticky ends and used to extend monomer in ICA. Monomer C (this part) can bind to Monomer A (K2066002) or the terminator (see WMiGEM2016 wiki. This part contains the following: UNS 2 (WM standard gibson/amplification primer site) – UNS 4 (used to provide orthogonal amplification of monomers alone) – BsmBI site – sticky end 3 - Tet Repeat –8 bp spacer (taken as first 8 bp from UNS X) – BsmBI site – Sticky end 1 - UNS 5 (orthogonal amplification in conjunction with UNS 4) – UNS 3 (WM standard gibson/amplification primer site).

ICA method and sequence design based on Briggs et al., 2012 and its supplement.

Briggs, A. W., Rios, X., Chari, R., Yang, L., Zhang, F., Mali, P., & Church, G. M. (2012). Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic acids research, gks624.

The UNS2, UNS3, UNS 4, and UNS 5 sequences are taken from Torella et al. 2013 and are ideal for cloning long sequences of monomers.

Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, gkt860.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 107