Part:BBa_K4335009

Insert site+gRNA scaffold

The transcript of gRNA scaffold can bind to SpCas9. The Insert site can be inserted into the sgRNA targeting specific target genes through the Golden Gate assembly.

Usage

Template of sgRNA. Use Golden Gate (BsaⅠ) to replace the template sequence with a ~20bp guide sequence. Add 'ACTT' on the 5'-terminal of your guide sequence and 'AAAC' on the reverse 5'-terminal.

sgRNA

Result

The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.

Gel run of samples from colony PCR.Primer length ~660 bp. M: 2000 bp DNA Marker; 38: pTX2038 vector; 40: pTX2040 vector; sgRNA1-3: different sgRNAs is designed.

Figure 1 shows that the colony PCR has successfully amplified about 660 bp bands, which correspond to the vector corresponding to the sgRNA of each target gene (pTX2038/pTX2040). It preliminarily indicates that we have successfully inserted the sgRNA of the target gene into the vector (pTX2038/pTX2040), and then Sanger sequencing of each insert site has been carried out, which proves that we have successfully constructed.

collection

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 site found at 25
  Illegal BsaI.rc site found at 7