The plasmids used for overexpression of Sst2 or Kel1 by the ADH1 promoter were constructed using the NEB Gibson Assembly Cloning Kit (E2611S; NEB) as advised by the manufacturer’s instructions. All plasmids were built using the pRSII416 vector backbone (Plasmid #: 35456; Addgene [Chee & Haase, 2012 (link)]). The vector backbone was linearized with SacI-HF (R3156S; NEB) and ApaI (R0114S; NEB) restriction enzymes before Gibson assembly. Primers were constructed using the online NEBuilder assembly tool (v2.6.0, https://nebuilder.neb.com/ ) and are listed in Table S3. The forward and reverse 3XFLAG sequences were obtained from p3xFLAG-CMV-14 and synthesized as oligos for PCR amplification with primers in Table S3. The 1 kilobase of DNA upstream of the ADH1 was amplified from genomic DNA to provide the ADH1 promoter.
Neb gibson assembly cloning kit
Manufactured by New England Biolabs
The NEB Gibson Assembly Cloning Kit is a DNA assembly method developed by New England Biolabs. It enables the efficient and seamless joining of multiple DNA fragments in a single reaction. The kit provides the necessary reagents and protocols to perform this DNA assembly process.
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2 protocols using neb gibson assembly cloning kit
1
Overexpression of Sst2 and Kel1 in Yeast
2
Cloning and Validation of pEVS143 Plasmids
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pEVS143 was amplified using pEVS143_expF and pEVS143_expR primers. Gene ORFs were amplified from MJM1100 genomic DNA using gene-specific forward and reverse primers. Amplified gene inserts were assembled with amplified pEVS143 by Gibson assembly using NEB Gibson Assembly Cloning Kit (NEB) or NEBuilder HiFi DNA Assembly Master Mix (NEB). The assembly reactions were transformed into chemically competent NEB5α or DH5α λpir cells and candidate transformants were selected using kanamycin. Each plasmid was screened by PCR using primers MJM-738F and MJM739R; M13 −48 rev and MJM-739R; or MJM-738F and the respective assembly reverse primer, MJM-739R and the assembly forward primer, and/or MJM-738F and MJM739R. To construct pEVS143-VF_A1014, VF_A1014 was synthesized with flanking AvrII and BamHI restriction sites and cloned into pEVS143 by GenScript (Piscataway, NJ) and plasmid was confirmed by whole plasmid sequencing. pEVS143-VF_A0506 was confirmed by Sanger sequencing using primers M13 −48 rev, RYI225, RYI226, RYI227, RYI228, RYI229, RYI230, and MRH049. Remaining plasmids were confirmed by Sanger sequencing using primers MJM-738F and MJM-739R; MJM-738F and the respective assembly reverse primer and/or MJM-739R and the assembly forward primer; or pEVS143_seqF and pEVS143_seqR.
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