pATDt
(Plasmid #249499)

• Purpose: Expresses tdTomato – Monodirectional chloroplast expression vector (psbH Rescue) in Chlamydomonas Reinhardtii photosynthetically deficient strain CC4388
• Depositing Lab: Isabel Desgagné-Penix
• Publication: Engineering bidirectional chloroplast promoters for tunable co-expression of multiple genes in microalgae (unpublished)

Backbone

• Vector backbone: pASAPI
• Backbone manufacturer: Chlamydomonas Resource Center; http://www.chlamycollection.org
• Backbone size w/o insert (bp): 6333
• Total vector size (bp): 7752
• Modifications to backbone: The 5′ untranslated region (5′ UTR) and promotor region of the psbD gene was added
• Vector type: Bacterial Expression ; Chlamydomonas reinhardtii chloroplast genome
• Selectable markers: Photosynthesis-based selection by restore of the psbH gene

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: NEB Stable E. coli strain can also be used
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: tdTomato (red fluorescence)
• Alt name: tdTomato
• Species: Discosoma sp.
• Insert Size (bp): 1431
• Mutation: tdTomato résulte de la fusion de deux copies du gène dTomato, une version optimisée de la protéine fluorescente rouge DsRed issue du corail Discosoma sp.
• GenBank ID: GenBank: AAV52169 GenBank: AAV52169
• Promoter: The psbD/ 5′ UTR, a light-activated chloroplast promoter enabling expression of the endogenous psbD gene

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: complete plasmid sequencing
• 3′ sequencing primer: complete plasmid sequencing

Resource Information

• Supplemental Documents:
  • pATDt.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The plasmid pATDt is designed for expression of one transgene in the chloroplast of Chlamydomonas reinhardtii using the monodirectional promoter psbD/ 5′ UTR. This vector is intended for use in the C. reinhardtii strain CC4388, which is photosynthetically deficient due to disruption of the psbH gene, encoding a small subunit of Photosystem II. Correct integration of the plasmid into the chloroplast genome restores psbH function and rescues photosynthesis. Selection is therefore performed by photoautotrophic growth, without the need for antibiotic selection.