pAT253
(Plasmid #40943)

• Depositing Lab: Angela Taddei
• Publication: Dubarry et al Genes Dev. 2011 Jul 1;25(13):1365-70.

Backbone

• Vector backbone: pAFS135
• Backbone manufacturer: Andrew W. Murray Lab, UCSF (Straight, Sedat, & Murray, 1998. PMID: 9813090)
• Backbone size w/o insert (bp): 4600
• Total vector size (bp): 6433
• Vector type: Yeast Expression
• Selectable markers: HIS3

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: GFP-LacI**
• Alt name: GFP-LacI
• Alt name: LacI2G(4mutations)
• Alt name: LacI
• Species: E. coli
• Insert Size (bp): 1850
• Mutation: LacI**/LacI2G(4 mutations) (see comment below); lacI-I12 mutation (P3Y); C-terminal 11 amino acids deleted to prevent tetramerization
• Promoter: His3 promoter fragment
• Tags / Fusion Proteins:
  • GFP(S65T, V163A) (N terminal on insert)
  • SV40 NLS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XhoI (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: GFP-F, M13_pUC_rev
• 3′ sequencing primer: M13_pUC_fwd

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

The plasmid pAFS135 (Straight, Sedat, and Murray, 1998) contains GFP(S65T, V163A)-LacI(P3Y, del C-terminal 11 aa)-SV40 NLS under the control of a HIS3 promoter fragment. To construct pAT123, the HIS3 marker of AFS135 was replaced with a ScaI-DraIII fragment from RS405 containing LEU2 (a kind gift from Karine Dubrana and Susan Gasser).

The plasmid pAT222 (Addgene plasmid #40942) was obtained by site-directed mutagenesis of pAT123 to create GFP-LacI**, using the primers (base mutations in lowercase):

5’- GTGGCACAgCAACTGGCGGaCAAAggtggcggaTCGTTGCTGATTGGCGTTGCCtCCT-3’ and
5’- AGGaGGCAACGCCAATCAGCAACGAtccgccaccTTTGtCCGCCAGTTGcTGTGCCAC- 3’.

The LacI** variant was then subcloned into pAFS135 using NotI and XhoI restriction sites to create plasmid pAT253.