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pAtCHYb1Δ129N
(Plasmid #53367)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 53367 Standard format: Plasmid sent in bacteria as agar stab 1 $75

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pTrcHis A
  • Backbone manufacturer
    Invitrogen
  • Backbone size w/o insert (bp) 4414
  • Total vector size (bp) 5062
  • Modifications to backbone
    A cDNA encoding an Arabidopsis thaliana beta-carotene hydroxylase was excised from the original cDNA library plasmid [see Sun Z, Gantt E, Cunningham FX Jr (1996) Cloning and functional analysis of the beta-carotene hydroxylase of Arabidopsis thaliana. J Biol Chem. 271, 24349-24352.] with PvuII (within the cloning vector upstream of the 5’ end of the cDNA) and NotI (within the adaptor at the 3’ end of the cDNA), blunted using the Klenow enzyme, and ligated in the forward orientation in pTrcHis A that had been digested with BamHI and blunted with the Klenow enzyme. The resulting plasmid, which retained a BamHI site immediately upstream of the cDNA, was digested with BamHI, then partially digested with BglII (for which there was one site within the open reading frame of the cDNA and a second site in the vector immediately after the cDNA), and a polynucleotide of ca. 5 kB was gel purified and self-ligated. The resulting plasmid contains a truncated open reading frame (lacking codons for the first 129 amino acids) for the A. thaliana beta-carotene hydroxylase, fused at the N terminus to two codons that specify the amino acids MG and lying immediately downstream of and partially overlapping (but not fused to) a small open reading frame under the control of the strong Trc promoter. This plasmid is also known as pAtCHYb-BglII [Cunningham FX Jr, Gantt E (2007) A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynth Res. 92, 245-259.].
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    XL1 Blue
  • Growth instructions
    Can be used with pAC-BETA or pAC-BETAipi in E. coli strain TOP10 to produce beta-cryptoxanthin. For this purpose, grow liquid cultures on a platform shaker at 28 degrees Celsius for 2-3 days in darkness, or grow on agar plates at room temperature for 3-7 days.
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    chyB1
  • Alt name
    beta-carotene hydroxylase 1
  • Species
    A. thaliana (mustard weed)
  • Insert Size (bp)
    622
  • Mutation
    Deleted codons for the first 129 amino acids
  • GenBank ID
    U58919.1
  • Promoter Trc

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site BamHI (destroyed during cloning)
  • 3′ cloning site BamHI (destroyed during cloning)
  • 5′ sequencing primer pTrcHis-F
  • 3′ sequencing primer pTrcHis-R
  • (Common Sequencing Primers)

Resource Information

  • Terms and Licenses
  • Industry Terms
    • Not Available to Industry
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:

    pAtCHYb1Δ129N was a gift from Francis X Cunningham Jr (Addgene plasmid # 53367 ; http://n2t.net/addgene:53367 ; RRID:Addgene_53367)
  • For your References section:

    A study in scarlet: enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis. Cunningham FX Jr, Gantt E. Plant J. 2005 Feb;41(3):478-92. 10.1111/j.1365-313X.2004.02309.x PubMed 15659105