Part:BBa_K1433009

attL-RTerminator-RTerminator-attR-B

This part is a standard is adding the next bi-terminator unit into GENE SOCKET together with current insertion parts, which is a crucial design for continuously circuit construction. Besides, since the circuit situation can be quite different, we provide two parts to help you deal with most cases: BBa_K1223009 and BBa_K1223010.

Composition

1. attL and attR sites: Recognition sites for Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase.
2. Terminator(reverse): Unidirectional terminator.
3. Homologous arm: Homologous arm B.

Matters need attention

• Homology region of inserted part: The homology regions flanking beside inserted parts are added by PCR. The Latter parts must have SpeI restriction site to link with our standard parts.



• Restriction sites：The inserted part is cut by SpeI after PCR and standard parts are cut by XbaI and PstI. After ligation, no restriction site exists between the inserted part and the standard part.
  • SepI and XbaI are isocaudomer.
• Bi-terminator regions：The bi-terminator region is inverted in standard parts. The terminator we choose here is a unidirectional terminator. It only works after Int and Xis flipping over the attL/attR site.



• The last homology region：The last homology region is a special site which will not change during circuit construction.
• Additional promoter：If the inserted part has terminator, an additional promoter should be added behind it to allow Int expression. This promoter here is BBa_J23110. You can also change the promoter or use you own promoter. See Solution page for more information.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 24
  Illegal BsaI.rc site found at 282