Part:BBa_K5399008

pAtU6-sgRNA-SBEI-3xFLAG-SV40 NLS-cas9-NLS

This component includes pAtU6, sgRNA-SBEI, 3xFLAG, SV40 NLS, Cas9, and NLS. The pAtU6 is used to initiate the transcription of sgRNA-SBEI; 3xFLAG is used for the detection and purification of the Cas9 protein; SV40 NLS and NLS are used to target the Cas9 protein to the nucleus for the purpose of cleaving the SBEI gene.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1640
  Illegal PstI site found at 3062
  Illegal PstI site found at 3266
  Illegal PstI site found at 3296
  Illegal PstI site found at 4508
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1640
  Illegal PstI site found at 3062
  Illegal PstI site found at 3266
  Illegal PstI site found at 3296
  Illegal PstI site found at 4508
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1101
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1640
  Illegal PstI site found at 3062
  Illegal PstI site found at 3266
  Illegal PstI site found at 3296
  Illegal PstI site found at 4508
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1640
  Illegal PstI site found at 3062
  Illegal PstI site found at 3266
  Illegal PstI site found at 3296
  Illegal PstI site found at 4508
  Illegal NgoMIV site found at 1928
  Illegal NgoMIV site found at 3032
  Illegal NgoMIV site found at 3105
  Illegal NgoMIV site found at 3590
  Illegal NgoMIV site found at 4499
  Illegal NgoMIV site found at 4968
  Illegal NgoMIV site found at 4987
  
• 1000
  COMPATIBLE WITH RFC[1000]

a. Vector design and construct

We designed a pair of sgRNA sequences targeting the knockout of the SBEI genes using online sgRNA design tools. To prepare the sgRNAs, we first denatured the secondary structures of the complementary oligonucleotide strands by heating them, then gradually cooled the strands to allow annealing and formation of double-stranded sgRNAs under T4 Polynucleotide Kinase (New England Biolabs, UK). The vector psgR-Cas9-At was linearized using BbsI (FastDigest, Thermo Fisher, Waltham, MA, USA). The double-stranded sgRNAs were then ligated to the linearized vector using Quick T4 DNA ligase (New England Biolabs, UK) (Figure 1). The recombinant vectors psgR-Cas9-IbSBEI-sgRNA were subsequently transformed into E. coli (Figure 2).

Figure 1. plasmid map of psgR-Cas9-IbSBEI-sgRNA (BBa_K5399008).

Figure 2. Construction of psgR-Cas9-IbSBEI-sgRNA (BBa_K5399008). Left: E. coli with psgR-Cas9-IbSBEI-sgRNA; right: PCR product of IbSBEI-sgRNA (primer F: 5’-TGTAAAACGACGGCCAGT-3’; primer R: 5’-CTCCCGATAGGTGATACCTG-3’).