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Fig. 4 | Bioresources and Bioprocessing

Fig. 4

From: Self-cloning CRISPR/Cpf1 facilitated genome editing in Saccharomyces cerevisiae

Fig. 4

scCRISPR/Cpf1 facilitated tripleplex genomic integration of in vivo assembled DNA parts in S. cerevisiae. a scCRISPR/Cpf1 facilitated tripleplex genomic integration of β-carotene synthetic pathway of the crtI, crtYB, and tHMG1-crtE into the target sites Gal1-7, Gal80, and HO, respectively. b Efficiency of tripleplex genomic integration assisted by scCRISPR/Cpf1. c Pictures of transformants obtained by co-transforming donor DNA of β-carotene synthetic pathway with pSC-Pal and cr-Gal17/Gal80/HO. d PCR result of 1 white transformant (−) and 6 orange transformants (1–6) using primer pairs SQ1/SQ2, SQ3/SQ4, and SQ5/SQ6 for integration of crtI at the Gal1-7 (up), crtYB at the Gal80 (middle), and tHMG1-crtE at the HO (bottom), respectively. The horizontal red arrows indicate the PCR bands of correct integration. M: DNA Ladder 5000 or DNA Ladder 10,000. e HPLC analyses of β-carotene synthesis to evaluate the tripleplex genomic integration. β-carotene: β-carotene standard; BY4741: starting yeast strain; Colony1, Colony2, and Colony3: three orange colonies randomly selected. A, B, E, F, H, and K in orange rectangle: 50 bp homologous connector sequences. PTEF1, PHXT7, PGal1, and PGal10: promoters of the TEF1, HXT7, Gal1, and Gal10, respectively; TTPS1, TPGK1, TCYC1, and TADH1: terminators of the TPS1, PGK1, CYC1, and ADH1, respectively

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