pGFP-IBGFTet
(Plasmid #33356)

• Depositing Lab: Doug Mortlock
• Publication: Chandler et al Dev Biol. 2009 Mar 15;327(2):590-602. Epub 2009 Jan 3.

Backbone

• Vector backbone: pIBG-FTET
• Backbone manufacturer: Addgene (#33361)
• Vector type: Mouse Targeting
• Selectable markers: Neomycin (select with G418) ; tetracycline

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH10B
• Growth instructions: Do not select for this plasmid with tetracycline, as we have found that the Tet resistance cassette does not work reliably in high-copy plasmids such as this one. The Tet cassette MUST be transferred to a low-copy vector, e.g. a BAC, to allow Tet resistance in a reliable fashion. In BAC clones it has been extremely reliable.
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: EGFP-IRES-Bgeo + TetR

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: none (unknown if destroyed)
• 3′ cloning site: none (unknown if destroyed)
• 5′ sequencing primer: EGFP-C
• 3′ sequencing primer: LacZ-R

Resource Information

• Supplemental Documents:
  • pGFP-IBGFTet map
  • pGFP-IBGFTet, Primer list
  • pGFP-IBGFTet, Genbank file

Terms and Licenses

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

Depositor Comments

To generate the GFP-IRES-β-geo cassette, a PacI site was inserted was inserted upstream of the Kozak consensus of EGFP in pEGFP-C1 (kind gift of David Piston) and a PacI/XhoI fragment containing the EGFP open reading frame was inserted into pIBG-FTET (Chandler et al., 2007b) to create pGFP-IBGFTet (also known as pGIBGFTET).

A mismatch between author sequence and Addgene sequencing results was found at nucleotide position 5695 (with SV40pA-R primer) as well as at position 6913 (with EBV-rev primer). These sequence differences predate the construction of this plasmid.