pY2F
(Plasmid #53285)

• Purpose: Contains an Arabidopsis thaliana lcyE cDNA and produces an active lycopene epsilon-cyclase enzyme in E. coli. Use with pAC-BETA-At or pAC-BETAipi-At to produce alpha-carotene in E. coli.
• Depositing Lab: Francis X Cunningham Jr
• Publication: Cunningham FX et al Photosynth Res. 2007 May;92(2):245-59. Epub 2007 Jul 17.

Backbone

• Vector backbone: pBluescript SK-
• Backbone manufacturer: Stratagene
• Backbone size w/o insert (bp): 2958
• Total vector size (bp): 4884
• Modifications to backbone: A cDNA library plasmid referred to as y2 [Cunningham,F.X. Jr., Pogson,B., Sun,Z., McDonald,K.A., DellaPenna,D. and Gantt,E. (1996) Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8, 1613-1626.] contained an Arabidopsis thaliana lcyE cDNA in the reverse orientation relative to the lac promoter of the cloning vector (pBluescript SK-). To improve expression, the cDNA was excised from plasmid y2 as an XhoI-SmaI fragment of 1.9 kB and ligated in pBluescript SK- that had been digested with XhoI, blunted with the Klenow enzyme and then digested with SalI. The resulting plasmid contained the cDNA in the forward orientation relative to the lac promoter.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Growth instructions: When using together with plasmid pAC-BETA or pAC-BETAipi to produce alpha-carotene, grow liquid cultures at 28 degrees Celsius in darkness for 2-3 days , or grow on agar plates at room temperature for 3-7 days.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: lcyE
• Alt name: lycopene epsilon-cyclase
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 1929
• GenBank ID: U50738.1
• Entrez Gene: LUT2 (a.k.a. AT5G57030, LUTEIN DEFICIENT 2, LYCOPENE EPSILON-CYCLASE)
• Promoter: lac

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SalI (destroyed during cloning)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: M13pUC-rev
• 3′ sequencing primer: M13pUC-fwd

Terms and Licenses

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