Plasmids for comparing dual cassette and P2A-linked antibiotic resistance gene expression were based on pDM1203 [15 (link)]. Note that as a result, all the plasmids for this set of experiments use different actin15 promoter and actin8 terminator sequences derived from this parent plasmid. To make the dual cassette plasmid pDMDC a15-mNG coaA-HygroR, a codon-optimized mNeonGreen was first cloned into the BglII/SpeI cloning site of pDM1203 and the G418-resistance cassette was swapped for a hygromycin-resistance gene by restriction digest with NheI/NotI and Gibson assembly. The hygromygin resistance fragment was obtained using pDM1501 [15 (link)] as a PCR template. In contrast to all other plasmids in this study, the resulting dual cassette plasmid is bidirectional, with the antibiotic resistance gene encoded in one direction on the plasmid and the mNeonGreen encoded in the other direction. To make the P2A-linked plasmid pDMP2A a15-mNG-P2A-HygroR, the resistance cassette was removed from pDM1203 by XhoI/BamHI digest followed by blunt-ending and religating the resultant plasmid. A codon-optimized mNeonGreen was then inserted into the BglII/SpeI site along with P2A-HygroR by Gibson assembly. The expression cassettes of the generated plasmids were verified by Sanger sequencing with GENEWIZ (Azenta Life Sciences).
Comparing 2A Peptides and Antibiotic Resistance in D. discoideum
Plasmids for comparing dual cassette and P2A-linked antibiotic resistance gene expression were based on pDM1203 [15 (link)]. Note that as a result, all the plasmids for this set of experiments use different actin15 promoter and actin8 terminator sequences derived from this parent plasmid. To make the dual cassette plasmid pDMDC a15-mNG coaA-HygroR, a codon-optimized mNeonGreen was first cloned into the BglII/SpeI cloning site of pDM1203 and the G418-resistance cassette was swapped for a hygromycin-resistance gene by restriction digest with NheI/NotI and Gibson assembly. The hygromygin resistance fragment was obtained using pDM1501 [15 (link)] as a PCR template. In contrast to all other plasmids in this study, the resulting dual cassette plasmid is bidirectional, with the antibiotic resistance gene encoded in one direction on the plasmid and the mNeonGreen encoded in the other direction. To make the P2A-linked plasmid pDMP2A a15-mNG-P2A-HygroR, the resistance cassette was removed from pDM1203 by XhoI/BamHI digest followed by blunt-ending and religating the resultant plasmid. A codon-optimized mNeonGreen was then inserted into the BglII/SpeI site along with P2A-HygroR by Gibson assembly. The expression cassettes of the generated plasmids were verified by Sanger sequencing with GENEWIZ (Azenta Life Sciences).
Corresponding Organization : Boston University
Variable analysis
- 2A peptides used for comparison (e.g., P2A, T2A, F2A, etc.)
- Fluorescent protein expression (mNeonGreen, mCherry, mScarlet-I)
- Antibiotic resistance gene expression (G418, Hygromycin)
- Codon optimization for D. discoideum expression
- Transcriptional unit assembly (actin15 promoter, actin8 terminator)
- Backbone with D. discoideum origin of replication
- Not explicitly mentioned
- Not explicitly mentioned
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