pXZ13lacZalphaKanR
(Plasmid #160230)

• Purpose: (Empty Backbone) Like pXZ13lacZalpha, but adds second level of selection with Kanamycin resistance.
• Depositing Lab: Ian Duncan
• Publication: Modified donor plasmids to enhance Golden Gate assembly of Drosophila two stage Crispr/Cas9 cassette exchange (unpublished)

Backbone

• Vector backbone: pXZ13lacZalpha
• Backbone manufacturer: Dianne Duncan
• Backbone size (bp): 1961
• Modifications to backbone: Ampicillin resistance gene in pXZ13lacZalpha was replaced with Kanamycin gene from pET28a. Esp3I in Kanamycin gene was removed by overlap extension prior to insertion into pXZ13lacZalpha to render final construct usable in Golden Gate reactions.
• Vector type: Bacterial Expression, CRISPR
• Promoter: lac

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Cloning Information

• Cloning method: Gibson Cloning

Terms and Licenses

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

Depositor Comments

Modification of pXZ13lacZalpha construct to exchange Kanamycin gene for original Ampicillin gene. This improves upon the cloning efficiency of the original pXZ13 vector (reference below) in three ways: 1) The distance between the Esp3I sites is increased to increase cutting efficiency, 2) lacZalpha complementation is introduced to allow for blue/white screening of recombinants, and 3) Kanamycin selection removes 3 of the 4 original parental plasmids from forming colonies in the final plating.
Original vector reference:
Zhang X, Koolhaas WH, Schnorrer F. A Versatile Two-Step CRISPR- and RMCE-Based Strategy for Efficient Genome Engineering in Drosophila. G3 (Bethesda). 2014 Oct 15;4(12):2409-18