pAS1NB c Rosella I
Purposea biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Cytosolic localization.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||71245||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
- Total vector size (bp) 9040
Vector typeYeast Expression
Growth in Bacteria
Alt namesuper ecliptic pHluorin (SEP)
- Promoter PGK1
- Cloning method Restriction Enzyme
- 5′ cloning site BamHI (not destroyed)
- 3′ cloning site NotI (not destroyed)
- 5′ sequencing primer PPGK1-F (cagcctgttctcacacactc)
- 3′ sequencing primer yPGK1-term-R (AGCGTAAAGGATGGGGAAAG) (Common Sequencing Primers)
The coding sequence of DsRed.T3 was amplified from pQE31‑DsRed.T326 using the primer pair DsRedTUp2 (5'TAGGATCCCCACTAGTCGCCACCATGGCC TCCTC) and DsRedTDo (5'ATAGTTTAGCGGCCGCTCAGT GATCAGACAGGAACAGGTGGTGG) designed to incorporate 5' BamHI and nested 3' BclI and NotI sites. Following digestion with BamHI and NotI, the amplified gene cassette was subcloned into the multi‑copy yeast expression vector pAS1NB under control of the PGK1 promoter and the resultant vector named pAS1NB‑DsRed.T3. The super ecliptic pHluorin (SEP) gene was amplified from the template pGEX‑2T‑SEP27 using the primer pair PHUp1 (5'GGGATCCTGGTCTTCGGGTGGAAGTAAAGGAG) and PHDo1 (5'‑ATAGTTTAGCGGCCGCTCAGTGATCAGATT TGTATAGTTCATCC) designed to incorporate flanking 5' BamHI and 3' NotI sites. The SEP gene cassette recovered by PCR was digested with BamHI and NotI, and cloned into pAS1NB‑DsRed.T3 digested with BclI and NotI to produce this vector. The expression product from this vector, named the Rosella biosensor, comprises DsRed.T3 fused to SEP with an intervening linker of 9 amino acids (DPWSSSGDL).
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pAS1NB c Rosella I was a gift from Mark Prescott (Addgene plasmid # 71245 ; http://n2t.net/addgene:71245 ; RRID:Addgene_71245)
For your References section:Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast. Rosado CJ, Mijaljica D, Hatzinisiriou I, Prescott M, Devenish RJ. Autophagy. 2008 Feb;4(2):205-13. Epub 2007 Nov 21. 5331 [pii] PubMed 18094608