Afuco™ Platform
GTACATTATCAGTGTGCTCTCCACTTCTCCCCAGAGTCCATGTCAGACGCACTGACAAAGTGGGAACAGAAGCAGCCTTCCATCCC ATTGAGGAATACATGGTACACGTTGAAGAACATTTTCAGCTTCTCGAACGCAGAATGAAAGTGGATAAAAAAAGAGTGTATCTGGCC ACTGATGACCCTTCTTTGTTAAAGGAGGCAAAGACAAAGTAAGTTAGACCAACAAGTGGTTCTGTATGGGATTATCTCTTAGTTGAA GAAAATCCTTAATTCTGGGAACTTGTGGTTCTTGTTGCTAACTAATAGGTTCCAAAATCAAAGACTACATGTGCAAATATTAATCTAAT CAAGTCATACCTTACTAGCTGTATCTGATGCAAATTAAGAAGTCTAAAATGAATTAGACTGCTGATTTGTGTAGCATCACTAG Blue highlighted sequence: target site 1 Green highlighted sequence: target site 2 Red highlighted sequence: PAM Fut8-gRNA1: TCTCGAACGCAGAATGAAAGAGG Fut8-gRNA2: TGATGACCCTTCTTTGTTAATGG 2)
Construct the knockout vector Construct information
Fig 1. pGK1.1 vector map
Lane 1: undigested vector Lane M: DNA marker Lane 2-4: linearized vectors
Fig 2. pGK1.1 vector after BbsI digestion
Add joint sequence in gRNA To clone gRNA into the BbsI-linearized vector, the joint sequence is needed. A “CACC” was added into the 5’ of the sense strand and
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