Part:BBa_K4041004

Composite vibrio CDS template (TDH-vcgC-ctxA)

This is a non-coding part that consist of the TDH gene of the Vibrio Parahaemolyticus, vcgC gene of Vibrio vulnificus and the ctxA gene of Vibrio Cholerae. The genome template is used for cloning into a plasmid and contains conserved regions so teams could design detection kit based on the conserved regions.

The genes is one of the central and important gene that makes the Vibrios pathogenic. While it is important for its cytotoxicity, it itself could be served as a biomarker for the strains.

The following qPCR primer were proved to work in this block in in Vibrio
TDH Reverse	GACACCGCTGCCATTGTATAGT
TDH Forward	CCCGGTTCTGATGAGATATTGT
vcgC Reverse	CGCCAATAGCCTGTTCAGAT
vcgC Forward	GCAGTTCAAACATGGTCTCAAA
ctxA Reverse	GGTCTTATGCCAAGAGGACAGA
ctxA Forward	CCATCATCGTGCCTAACAAATC
The following RPA primer were proved to work in this block in in Vibrio
TDH Reverse	CTTATAGCCAGACACCGCTGCCATTGTATAGT
TDH Forward	CCCGGTTCTGATGAGATATTGTTTGTTGTTCG
vcgC Reverse	GCGAGTAGTGAGCCGATCCCGCCAATAGCCTG
vcgC Forward	GGCGCAGTTCAAACATGGTCTCAAAAAGGAGC
ctxA Reverse	GCAGTCAGGTGGTCTTATGCCAAGAGGACAGA
ctxA Forward	ACATATCCATCATCGTGCCTAACAAATCCCGT
The following crRNA were proved to work in this block in in Vibrio
TDH forward (crRNA1)	ACTGTGAACATTAATGATAA
vcgC forward (crRNA3)	ATAGCACTAATGTGTCATCT
vcgC reverse (crRNA4, with PAM)	GCGGAGACGAGATCGCTATC
ctxA Forward (crRNA5)	TGATCATGCAAGAGGAACTC
crRNA 2 (high background) and crRNA 6 (no cleavage signal) is not functional from design, and is discarded	TGATCATGCAAGAGGAACTC

<img style = "width: 90%"src= "" alt = "POC first table">

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 295
  Illegal BsaI.rc site found at 477