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Addgene

pAC-ZETAipi
(Plasmid #53284)

Ordering

This material is available to academics and nonprofits only.
Item Catalog # Description Quantity Price (USD)
Plasmid 53284 Standard format: Plasmid sent in bacteria as agar stab 1 $85

Backbone

  • Vector backbone
    pAC-EHER
  • Backbone manufacturer
    Francis X. Cunningham, Jr.
  • Backbone size w/o insert (bp) 13581
  • Total vector size (bp) 8352
  • Modifications to backbone
    Plasmid pAC-EHER was digested with DrdI and partially digested with DraIII, a 9.46 kB fragment was gel-purified, the ends were made blunt with the Klenow enzyme, and the fragment was ligated to give pAC-BETA-crtX. Plasmid pAC-ZETAipi was constructed by excising a ca. 2.25 kB PvuII–BamHI fragment containing the Synechococcus sp PCC7942 phytoene desaturase gene, pds (crtP), fused to the N terminal portion of the lacZ gene with promoter, from pPDSdel35 [Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J, Sandmann G (1991) Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch C. 46, 1045-1051.], blunting it with the Klenow enzyme, and ligating it together with a 6.1 kB BglII + Klenow-blunted-SphI fragment obtained from pAC-BETAcrtX (see above).
  • Vector type
    low copy number bacterial cloning vector

Growth in Bacteria

  • Bacterial Resistance(s)
    Chloramphenicol, 25 μg/mL
  • Growth Temperature
    30°C
  • Growth Strain(s)
    Top10
  • Growth instructions
    Grow liquid cultures on a platform shaker at 28 degrees Celsius in darkness for 2-3 days for best zeta-carotene production, or grow on agar plates at room temperature for 3-7 days. The pAC-ZETAipi plasmid produces a mixture of cis isomers of zeta-carotene in E. coli, predominantly 9,15,9′-tri-cis-zeta-carotene and 9,9′-di-cis-zeta-carotene [see Isaacson T, Ohad I, Beyer P, Hirschberg J (2004) Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants. Plant Physiol. 136, 4246-4255.]
  • Copy number
    Unknown

Gene/Insert

  • Gene/Insert name
    pds
  • Alt name
    crtP
  • Species
    Synechococcus PCC7942
  • Insert Size (bp)
    2249
  • GenBank ID
    X55289.1
  • Promoter lac

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site SphI (destroyed during cloning)
  • 3′ cloning site BglII (destroyed during cloning)
  • 5′ sequencing primer none
  • 3′ sequencing primer none
  • (Common Sequencing Primers)

Resource Information

  • A portion of this plasmid was derived from a plasmid made by
    Plasmid pPDSdel35 [Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J, Sandmann G (1991) Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch C. 46, 1045-1051.] was obtained from Dr. Joseph Hirschberg of the Hebrew University of Jerusalem.
  • Article Citing this Plasmid

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

For better yield of low copy number pAC-based plasmids, grow liquid cultures on a platform shaker at ca. 30 degrees Celsius. When cultures reach early stationary phase, dilute 2-fold with growth medium, add spectinomycin (150 mg/liter), and "amplify" for several hours before harvest. For plasmid selection and maintenance in E. coli, use chloramphenicol at 30 mg/liter.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pAC-ZETAipi was a gift from Francis X Cunningham Jr (Addgene plasmid # 53284 ; http://n2t.net/addgene:53284 ; RRID:Addgene_53284)
  • For your References section:

    A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Cunningham FX Jr, Gantt E. Photosynth Res. 2007 May;92(2):245-59. Epub 2007 Jul 17. 10.1007/s11120-007-9210-0 PubMed 17634749