Composite
Part:BBa_K3407025:Design
Designed by: Alicia Rodrguez Molina Group: iGEM20_TUDelft (2020-10-23)
pKDsgRNA_4.3 sgRNA targetting phage T7 gene 4.3
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The biobrick is constructed by two PCR’s on a commercially bought plasmid pKDsgRNA-p15 and a Gibson Assembly. The primers used were the following:
1rst PCR:
forward primer 5’ - tttataacctccttagagctcga - 3’ reverse primer 5’ - tgcatccactaaagttaccg gttttagagctagaaatagcaag - 3’
Second PCR:
forward primer 5’- ccaattgtccatattgcatca 3’ reverse primer 5’ - cggtaactttagtggatgcaggtgctcagtatctctatcactga - 3’.
The sequence of the protospacer is highlighted in bold.
A Gibson Assembly was done with both PCR fragments and the resulting plasmid was transformed in E. coli DH5alpha and recovered on LB with 60 ng/µl spectinomycin. The plasmid was confirmed by sequencing.