pgMAX system-EFGP

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	The PCR-amplified product was inserted into the EcoRV site of pgMAX and transfected in E. coli.

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The PCR-amplified product was inserted into the EcoRV site of pgMAX and transfected in E. coli.

Publication protocol

"Bicistronic expression vectors, pIRESpuro3 and pZero-2, were purchased from Thermo Fisher Scientific. DsRed2 and pEGFP-N1 plasmids were purchased from Clontech Laboratories (Palo Alto, CA, USA) [9]. The Bgeo gene (a combined gene of β-galactosidase and neomycin phosphotransferase) was kindly provided by Dr. Smith [10]. For plasmid construction, PCR-based mutagenesis was performed. The conditions for PCR with high-fidelity Pfu DNA polymerase (Agilent Technologies) were empirically modified (denaturation at 94°C for 20 s, an annealing step at the calculated temperature (ca. 50°C) for 30 s and an extension at 72°C for 30 s, for 35 cycles). Amplified PCR products were gel-purified with a gel extraction kit (Macherey-Nagel GmbH, Dueren, Germany). The inhibitory unit (iUnit) was PCR-amplified from pZero2 (Invitrogen, Carlsbad, CA, USA)) with a specific oligo DNA (iFor and iRev, see S1 Table). Additional restriction enzyme sites were introduced with oligonucleotide DNA primers (S1 Table), and protocols using the pgMAX system as well as the entire pgMAX sequence have been deposited in protocols.io. (DOI dx.doi.org/10.17504/protocols.io.zq3f5yn). The SwaI restriction site; lac promoter (lacP) and operator (lacO); Kozak sequence (Kozak), PmeI restriction site; BamHI site; Flag protein tag; BamHI site; and EcoRI, EcoRV and XhoI restriction sites were constructed using a PCR. The blunt-end product was inserted into the EcoRV site in pBluescriptIISK- (Agilent Technologies) using standard ligation techniques (Takara DNA Ligation kit ver.2.1, Takara, Otsu, Japan). For the transformation, XL10-Gold ultracompetent cells (Tetr Δ(mcrA)183 Δ(mcrCB‒hsdSMR‒mrr)173 endA1 supE44 thi‒1 recA1 gyrA96 relA1 lac Hte [F'proAB lacIqZΔM15 Tn10 (Tetr) Amy Camr] (Agilent Technologies) were used.

A blunt-end DsRed2 DNA fragment was amplified using Pfu DNA polymerase with DsRed2-specific oligo DNA (DsRed2for: AaaGCTAGCatgGCCTC CTCCGAGAAC GTCATCA; DsRed2rev: aaaGAATTCagatctcaggaacaggtggtg). A blunt-end enhanced green fluorescent protein (EGFP) DNA fragment was amplified using high-fidelity Pfu with EGFP-specific oligo DNA (EGFPfor: cccGCTAGCatgGTGAGCAAGGGCGAGGAG; EGFPrev: cccGGTACCGGCGGCGGTCACGAACTCCAG). The PCR-amplified product was inserted into the EcoRV site of pgMAX and transfected in E. coli."

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