Quickcut not 1

After liquid and plate culture, the plasmid library was extracted using a plasmid miniprep Kit (TIANGEN, #DP103). Then, QuickCut™ Not I (Takara, #1623) was used for fragments recovery. After gel recovery of the correct fragments using the Plus DNA Clean/Extraction Kit, the samples of the 509 oligos pool (1F, 3F and 5F) and the 11520 oligos pool (passage-1 and passage-5 of 1F and 3F) were sequenced directly. To obtain more complete information, we performed a PCR of the constructed plasmid to amplify the 11,520 oligos pool (passage-1 and passage-5 of 1F and 3F) using Q5® High-Fidelity DNA Polymerases and the primer pair F02/R02. The thermocycling protocol was: (1) 98 °C for 5 min, (2) 98 °C for 30 s, (3)54 °C for 30 s, (4) 72 °C for 10 s, then repeat steps 2–4 five times, followed by a final elongation at 72 °C for 5 min. The products were purified using the Plus DNA Clean/Extraction Kit (GMbiolab Co, Ltd. #DP034P) and sequenced.

Primers to amplify the full-length CUX1 coding sequence (CDS) were designed using Oligo 6 software based on the CUX1 sequence downloaded from NCBI. The full-length CDS was amplified using lamb skin tissue cDNA as a template. The fragment was cloned into the PEX-1 overexpression vector, and successful cloning was confirmed by double digestion with QuickCut™ XhoI (Takara, Japan) and QuickCut™ NotI (Takara, Japan). The overexpression vector were sent to the Tsingke Biotechnology Co., Ltd. (Nanjing, China) for verification.
The CUX1 primer sequences are as follows:
PEX-1-CUX1-F: CCCaagcttATGGCGGCCAATGTGGGATC
PEX-1-CUX1-R: CCGctcgagACACTGCCACAGGTCGCCG

All primers in this experiment were ordered from Comate Bioscience (Changchun, China). The fluorescent ssDNA reporter (5′6-FAM-TTATT-BHQ1-3′) and lateral flow strip test reporter (5′6-FAM-TTATT-Biotin-3′) were ordered from General Biol Co., Ltd. (Anhui, China). The recombinant plasmid Cas12a (huLbCpf1) was purchased from Addgene (Watertown, MA, USA). The HiScribe™ T7 Quick High Yield RNA Synthesis Kit and NEbuffer 3.1 were purchased from New England Biolabs (MA, USA). The miRNeasy Mini Kit was purchased from QIAGEN (Hilden, Germany). QuickCut NotI, QuickCut EcoRI, TaKaRa Taq and RNase inhibitor I were purchased from TaKaRa Bio Inc. (Dalian, China). HindII, MseI and RsaI were purchased from New England Biolabs (Ipswich, MA, USA). The TwistAmp™ basic kit was purchased from TwistDx Ltd. (Hertfordshire, UK). Cas12/13-specific nucleic acid detection kits were purchased from Tiosbio Inc. (Beijing, China). DNA genomes from eight pathogens (T. vaginalis, Candida albicans, Mycoplasma hominis, Neisseria gonorrhoeae, Escherichia coli, Cryptosporidium parvum, Giardia lamblia and Toxoplasma gondii) and the human genome were provided by our laboratory. The human vaginal secretion, semen and urine genome used for clinical sample testing were sourced from the Second Hospital of Jilin University in Changchun, Jilin Province.