pT2-eab2-[GTR]
(Plasmid #46144)

• Depositing Lab: Wenbiao Chen
• Publication: Boniface et al Genesis. 2009 Jul;47(7):484-91. doi: 10.1002/dvg.20526.

Backbone

• Vector backbone: pBluescript II KS+
• Backbone manufacturer: stratagene
• Total vector size (bp): 8831
• Vector type: Cre/Lox ; Tol2 zebrafish transgenic expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: EGFP/mCherry
• Alt name: EGFP
• Alt name: mCherry
• Insert Size (bp): 6500

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: KpnI (not destroyed)
• 3′ cloning site: SpeI (not destroyed)
• 5′ sequencing primer: M13R
• 3′ sequencing primer: M13F

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • National Institute of Genetics Limited Use Label License for Tol2 System
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This plasmid can be used to make a transgene that expresses EGFP but can be switched to express mCherry if inverted.

This reporter is controlled by a 2.3 kb regulatory sequence consisting of 0.2 kb Xenopus EF1alpha enhancer and 2.1 kb zebrafish
beta-actin 2 sequence including the 1.5 kb enhancer/promoter sequence, the 1st exon (86 bp) and part of the 1st intron (490 bp). To generate a complete intron, the splice acceptor from the rabbit beta-globin gene was placed upstream of the EGFP reporter and a synthetic splice acceptor was placed upstream of the mCherry reporter. The synthetic splice acceptor contains the zebrafish consensus sequence along with intronic and exonic splice enhancers.

There are some discrepancies between Addgene's and the depositor's sequence--these do not affect plasmid function.