Sources

Under construction 9/14/07.

Components

Here is the provenance of the components that we have used. For details of cloning, see the individual clone pages or the full methods paper.

Gateway components

The entry clones and destination vectors are all based on (three-insert) multisite Gateway technology from Invitrogen, specifically pDONR P4-P1R, pDONR221 (attP1-attP2), pDONR P2R-P3, and pDest R4-R3. Our understanding from Invitrogen is that they do not usually require any MTAs for clones generated by individual labs using their vectors. (You still need to buy recombination enzyme from them though!)

Sequence information all comes from the Invitrogen website. In some cases we have noticed point mutations in our constructs relative to their sequence. The most notable example is a point mutation in the ccdB coding sequence within the destination vector. In at least a couple of cases, we have seen a mutation in clones from different labs, leading us to believe that it derived from the original Invitrogen clone.

fluorescent proteins (FPs)

EGFP

EGFP is the enhanced green fluorescent protein (1).

mCherry

mCherry is the monomeric red fluorescent protein (2, 3).

Subcellular localization tags

nls-

The nuclear localization sequence is a 9 amino acid peptide (MAPKKKRKV) fused to the N-terminus of the fluorescent protein. The sequence was taken from pCS2+nls vector.

-CAAX

The CAAX box (SEQ) encodes a 21 amino acid fusion to the C-terminus of the fluorescent protein. The sequence is from human H-ras (last 21 amino acids) and results in prenylation of the protein, targeting it to the plasma membrane (4).

H2A-

H2A encodes the zebrafish histone variant H2A.F/Z, which has previously been shown to label chromatin when fused to EGFP (5). H2A.F/Z was cloned from cDNA, and fused to the N-terminus of mCherry.

References

(1) Zhang G, Gurtu V, Kain SR. 1996. An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochem Biophys Res Commun 227:707-711.

(2) Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567-1572.

(3) Gray NW, Weimer RM, Bureau I, Svoboda K. 2006. Rapid redistribution of synaptic PSD-95 in the neocortex in vivo. PLoS Biol 4:e370.

(4) Moriyoshi K, Richards LJ, Akazawa C, O'Leary DD, Nakanishi S. 1996. Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP. Neuron 16:255-260.