Mammalian DamID Plasmids

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.

pLgw CBX1-V5-EcoDam sequence
pLgw CBX1-V5-EcoDam map