Applications
- Construction of custom EZ-Tn5™ Transposons.
- EZ-Tn5 Transposons made with pMOD-3<R6Kγori/MCS> and
pMOD-5<R6Kγori/MCS> can be used for a variety of
rescue cloning applications.*
EPICENTRE offers five different EZ-Tn5™ pMOD™ Transposon Construction
Vectors* for the preparation of custom EZ-Tn5 Transposons (Table 1). Each
vector contains a multiple cloning site (MCS) flanked by the hyperactive
19-bp Mosaic Ends (ME, denoted by <MCS>) that are specifically and
uniquely recognized by EZ-Tn5 Transposase. To prepare the transposon, clone
any DNA sequence of interest (e.g., selectable marker, control element,
reporter gene) into the MCS and then generate the transposon either by
PCR amplification or restriction enzyme digestion. Any of the Transposon
Construction Vectors can be used to generate an EZ-Tn5 Transposon, but
they offer different features.
The Transposon Construction Vectors pMOD-2<MCS> and pMOD-3<R6Kγori/MCS> are
pUC-based vectors. They consist of ME sequences that flank an MCS in
a vector with a colE1 origin of replication (Fig. 1). EZ-Tn5 pMOD-3<R6Kγori/MCS> also
contains an R6Kγori within the ME sequences, which is useful
for a variety of rescue cloning applications. These vectors work well
for constructing transposons in most cases. However, if the transposon
is prepared by restriction enzyme digestion, there is a chance that the
uncut pMOD vector will contaminate the transposon constructed.
To solve this problem, the colE1 ori was eliminated in pMOD-4<MCS> and
pMOD-5<R6Kγori/MCS>, which only have the R6Kγori. Replication
from the R6Kγ origin in these two new vectors is dependent on the pir gene
product produced by TransforMAX™ EC100D™ pir+ and pir-116 E.coli cells.
Since most bacterial strains do not contain a pir gene, the uncut
plasmid DNA that contaminates these transposon preparations cannot replicate
and background problems are eliminated.
Benefits
- The MCS enables easy cloning of any DNA of interest.
- Hyperactive 19-bp Mosaic End sequences flanking the MCS for high-efficiency
transposition using EZ-Tn5 Transposase.
- Unique primer-binding sites at each end of the transposon for bidirectional
sequencing of the insertion site using the Forward and Reverse Sequencing
Primers (available separately). No need to design your own primers.
- Three options can be used to prepare an EZ-Tn5 Transposon–digestion
with Pvu II, digestion with PshA I, or PCR amplification
using the PCR primers provided with the vector.
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Figure 1 (click to enlarge). Features of the EZ-Tn5™ pMOD™ Transposon
Construction Vectors.
Figure 2 (click to enlarge). EZ-Tn5™ Transposon Construction
Vectors pMOD™-2<MCS> and pMOD™-3<R6Kγori/MCS> replicate
in standard E. coli strains using a colE1 origin of replication. Transposons
made with the pMOD™-3<R6Kγori/MCS> vector
also have an R6Kγori within the transposon, for rescue
cloning applications.
Figure 3 (click to enlarge). EZ-Tn5™ Transposon Construction
Vectors pMOD™-4<MCS> and pMOD™-5<R6Kγori/MCS> lack
a colE1 origin of replication and require E. coli strains that
produce a pir gene product for replication. This results
in less background from uncut plasmid DNA when an artificial transposon
is prepared by restriction endonuclease digestion. |
