CRISPR/Cas9 vector with visual protein fusion antibiotic selection marker as well as construction method and application of CRISPR/Cas9 vector
A technology for screening markers and construction methods, which is applied in the field of genetic engineering, can solve the problems of large transformation vectors and affect transformation efficiency, and achieve the effects of reducing size, improving transformation efficiency, and reducing workload
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[0028] This example provides a method for constructing a CRISPR / Cas9 vector with a visible protein fusion antibiotic selection marker, and the vector is used to construct a gene editing vector for the tomato SlD14 gene. plant.
[0029] 1. Construction of a CRISPR / Cas9 vector with a visible protein fusion antibiotic selection marker
[0030] S1, artificially synthesize the primer sequences shown in SEQ ID NO.2 and SEQ ID NO.3, take the 0380-GN carrier (gifted by Mr. Li Yi, University of Connecticut, USA) as the template, use The Max Super-Fidelity DNAPolymerase kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) was used to amplify the GUS::NPTII nucleotide sequence; artificially synthesized as shown in SEQ ID NO.4 and SEQ ID NO.5 Primer sequences, using pYLCRISPR-Cas9Pubi-H vector (gifted by Professor Liu Yaoguang of South China Agricultural University) as a template, using The Max Super-Fidelity DNAPolymerase kit (purchased from Nanjing Novizan Biotechnology Co.,...
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