pAC-VIOL
(Plasmid #53087)

• Purpose: Produces the carotenoid violaxanthin in E. coli.
• Depositing Lab: Francis X Cunningham Jr
• Publication: Neuman et al Plant J. 2014 Apr;78(1):80-93. doi: 10.1111/tpj.12451. Epub 2014 Mar 12.

Backbone

• Vector backbone: pAC-ZEAXipi
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 10040
• Total vector size (bp): 12814
• Modifications to backbone: An Arabidopsis thaliana cDNA encoding zeaxanthin epoxidase (zep) was modified so as to delete codons for the first 60 amino acids (the putative chloroplast transit sequence) and cloned in the NdeI and XhoI sites of pET30C so that expression was under the control of the T7 promoter. This plasmid, pET-AtZEP (unpublished), was a gift of Dr. Martin Sergeant, at that time at the University of Warwick. The zep gene with T7 promoter were excised from pET-AtZEP as a Klenow-blunted BspHI-BspHI fragment of ca. 2.4 kB and cloned in the Klenow-blunted AseI site of pAC-ZEAXipi. Insert orientation and sequences at the junctions of the insert were confirmed by DNA sequencing.
• 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 at 28 degrees Celsius for 2-3 days for best violaxanthin production, or grow on agar plates at room temperature for 3-7 days.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: zep
• Alt name: zeaxanthin epoxidase
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 2774
• Mutation: Lacks codons for the first 60 N-terminal amino acids that are thought to comprise the chloroplast transit sequence
• GenBank ID: AF281655.1
• Entrez Gene: ABA1 (a.k.a. AT5G67030, ABA DEFICIENT 1, ARABIDOPSIS THALIANA ABA DEFICIENT 1, ARABIDOPSIS THALIANA ZEAXANTHIN EPOXIDASE, ATABA1, ATZEP, IBS3, IMPAIRED IN BABA-INDUCED STERILITY 3, K8A10.10, K8A10_10, LOS6, LOW EXPRESSION OF OSMOTIC STRESS-RESPONSIVE GENES 6, NON-PHOTOCHEMICAL QUENCHING 2, NPQ2, ZEAXANTHIN EPOXIDASE, ZEP)
• Promoter: T7

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AseI (destroyed during cloning)
• 3′ cloning site: AseI (destroyed during cloning)
• 5′ sequencing primer: tagccgctaaagcaaacacc
• 3′ sequencing primer: gatggatcttcgaagcgaac

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Plasmid pET-AtZEP, containing a truncated Arabidopsis thaliana zep cDNA under the control of the T7 promoter, was a gift of Dr. Martin Sergeant, at that time of the University of Warwick.

Terms and Licenses

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

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.