Note: the most popular DamID plasmids are now available from Addgene
We have made two sets of vectors for DamID in mammalian cells: one set for conventional transfections, and one set for lentiviral transductions. These vectors differ somewhat in their design and applications. Read more about choosing the right vector.
Plasmids for conventional transfection (set A)
pIND(V5)EcoDam
This is an expression vector for E.coli Dam, tagged at its N-terminus with a V5 epitope tag. In front of this tag is a small multiple cloning site for the in-frame cloning of your favorite ORF. The Dam part carries its own start codon, so the vector without any additional insert can be used to express Dam only (the absolutely essential control experiment!). In this case Dam is not V5-tagged, because the V5 tag does not have its own start codon. At present we only have a vector for fusing Dam to the C-terminus of your favorite protein. From work in Drosophila we know that Dam doesn’t mind fusions at its other end, but we don’t have a pIND-derived vector for this yet.
pIND (V5) EcoDam sequence
pIND (V5) EcoDam map
pINDgw RFA-V5-EcoDam
As above, but this vector is Gateway compatible, which means that you need to use GateWay recombination cloning to create a Dam-fusion of your protein. Many ORFs are now available as GateWay entry vectors from several sources. Otherwise you should make your own GateWay entry vector first. Note that this vector can not be used to express Dam only.
pINDgw RFA-V5-EcoDam sequence
pINDgw RFA-V5-EcoDam map
pINDgw Cbx1 V5 EcoDam
This vector may be used as a positive control. It expresses the Cbx1-V5-Dam protein.
pINDgw Cbx1 V5 EcoDam sequence
pINDgw Cbx1 V5 EcoDam map
Plasmids for lentiviral infections (set B)
pLgw RFC1-V5-EcoDam & pLgw EcoDam-V5-RFC1
These vectors are Gateway-compatible destination vectors to create N- or C-terminal Dam-fusion proteins. The V5 epitope tag serves as a linker between Dam and your favorite protein.
| pLgw RFC1-V5-EcoDam sequence pLgw RFC1-V5-EcoDam map |
pLgw EcoDam-V5-RFC1 sequence pLgw EcoDam-V5-RFC1 map |
pLgw V5-EcoDam
This is the control vector for expression of V5-Dam only (the absolutely essential control experiment!).
pLgw V5-EcoDam sequence
pLgw V5-EcoDam map
pLgw Cbx1-V5-EcoDam
This vector may be used as a positive control. It expresses the Cbx1-V5-Dam protein.
