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Plasmids deposited by the Hegemann lab at the Chlamydomonas Resource Center

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de. Please verify your plasmids by sequencing.

 

pAphVIII (pPH75)

Streptomyces aminoglycoside-5 ́- phosphotransferase VIII (aphVIII) antibiotic selection marker gene for paromomycin selection, optimized to C. reinhardtii codon bias. map (GenBank; updated 09/2019)

 

pAphVII (pPH360)

Streptomyces aminoglycoside phosphotransferase (aphVII) marker gene for Hygromycin B selection, optimized to C. reinhardtii codon bias. map (GenBank)

 

For recombinant Cas9 protein expression in E. coli, we use plasmid pET-28b-Cas9-His (addgene #47327) of the Alex Schier Lab and their protocol for purification.

 

pHS-SpCas9 (pPH185)

Expression of Streptococcus pyogenes (Sp)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A heat-shock promoter. map

 

pHR-SpCas9 (pPH186)

Expression of Streptococcus pyogenes (Sp)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A/RBCS2 tandem promoter. map

 

pHS-SaCas9 (pPH187)

Expression of Staphylococcus aureus (Sa)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A heat-shock promoter. map

 

pHR-SaCas9 (pPH188)

Expression of Staphylococcus aureus (Sa)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A/RBCS2 tandem promoter. map

 

pCrU6.1-SpCas9 cloning (pPH195)

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #1. map

 

pCrU6.2-SpCas9 cloning (pPH158)

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #2. map

 

pCrU6.3-SpCas9 cloning (pPH159)

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #3. map

 

pCrU6.4-SpCas9 cloning (pPH160)

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #4. map

 

pCrU6.4-SpCas9 cloning/aphVIII (pPH340)

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map

 

pCrU6.4-SpPSY1/aphVIII (pPH332)

Vector for single guide RNA targeting PSY1 with RNA scaffold for Streptococcus pyogenes Cas9 (SpCas9), controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map

 

pCrU6.1-SaCas9 cloning (pPH193)

Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #1. map

 

pCrU6.2-SaCas9 cloning (pPH154)

Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #2. map

 

pCrU6.3-SaCas9 cloning (pPH155)

Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #3. map

 

pCrU6.4-SaCas9 cloning (pPH156)

Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #4. map

 

pCrU6.4-SaCas9 cloning/aphVIII (pPH339)

Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map

 

pCrU6.4-SaPSY1/aphVIII (pPH331)

Vector for guide RNA targeting PSY1 with scaffold for Staphylococcus aureus Cas9 (SaCas9), controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map

 


Reference

Greiner A, Kelterborn S, Evers H, Kreimer G, Sizova I, and Hegemann P. Targeting of photoreceptor genes via zinc-finger nucleases and CRISPR/Cas9 in Chlamydomonas reinhardtii. The Plant Cell October 2017. https://doi.org/10.1105/tpc.17.00659