Part:BBa_K2457004

Donor DNA for HDR into LacZ

BBa_K2457004 was engineered to be a building block of the standard BioBrick toolbox for bacterial genome: the ([http://2017.igem.org/Team:Amazonas_Brazil pCRISPeasy]). This BioBrick is the key part of the CRISPeasy-in (CRIN) framework, developed in 2017 by the Amazonas_Brazil team to make easier and accessible the genome editing based on CRISPR/Cas9. In this construction stays the gene of the template DNA that will be inserted in the edited genome. It's the first Registry vector for a Donor DNA.

Figure 1: BBa K2457004 circuit

Usage and Biology

Composed by the JK26 promoter, the coding sequence of the Green Fluorescent Protein (GFP) and the ilvBN terminator. It has 1810bp and was designed to contain two homologous arms with 300bp from the Operon Lac in the bacterial genome. Between the homologous arms, lies the GFP gene, flanked by NheI sites, allowing an RFC10 versatile one-step compatibility, the insertion of any other gene in GFP site. It has this structure especially for homology recognition by RecA enzyme which will break through the BioBrick to restore the cleaved sequence in the genome, having the aimed gene integrated.

Characterization

Figure 2:Sequencing electropherogram from BBa_K2457004

Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457004.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 321
  Illegal NheI site found at 1164
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]