Addgene - AAV-FLEX-rev-ChR2-tdtomato Plasmid Data

Plasmid 18917: AAV-FLEX-rev-ChR2-tdtomato

Gene/insert name:
Channelrhodopsin 2-tdtomato
Alt name:
ChR2
Insert size:
2367
Species:
Chlamydomonas reinhardtii
Fusion protein or tag:
tdtomato
Terminal:
C terminal on insert
Vector backbone:
AAV2
(Search Vector Database)
Vector type:
Cre/Lox ; Adeno-Associated Virus
Backbone size w/o insert (bp):
5340
Cloning site 5':
Spe1
Site destroyed during cloning:
Yes
Cloning site 3':
Spe1
Site destroyed during cloning:
Yes
5' sequencing primer:
CTGTGGCTGCGTGAAAGCCTTG List of Sequencing Primers
Bacterial resistance(s)
Ampicillin
Growth strain(s)
Stbl3
Growth temperature (℃):
30
Growth instructions:
Stbl2 E coli from Invitrogen Grow at 30 degrees in 2xYT media
High or low copy:
Low Copy
Person or lab that originally
cloned the gene/insert:
Karel Svoboda, Janelia Farm Research Campus
Sequence: View sequences (3)
Map:
View map
Supplemental document:
SmaI digest (application/pdf)
Supplemental document:
Notes from Addgene (2)
Principal Investigator:
Scott Sternson
Terms and Licenses:
MTA
Clontech Limited Use Label License

Comments:

These vectors are prone to recombination. This is a well known issue with these AAV vectors and is due to the inverted terminal repeats (ITRs) required for AAV production. To minimize recombination, we propagate these plasmids in Stbl2 cells from Invitrogen. Also, to minimize recombination, cells should be cultured at 30 C.

Note that these cultures will grow slowly (20 h for minipreps). Better yields and culture times are obtained with 2xYT as the media. This is strongly recommended.

Because recombination may still happen occasionally, we do a panel of restriction digestions to assess whether the ITRs are in tact. Separate digestions with PvuII, Sma1, and SnaB1 should be performed. The expected patterns can be calculated from the attached sequence. Please see Reviews in the right column for an image of Addgene's digest with these enzymes.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.

Article: A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. Atasoy et al (J Neurosci. 2008 Jul 9. 28(28):7025-30. PubMed)

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication. Also, please include the text "Addgene plasmid 18917" in your Materials and Methods section.