pMOD™ <MCS> Forward Sequencing Primer

pMOD™ <MCS> Forward Sequencing Primer


货号

MODFSP201


规格

1 nmole


存储温度

-20℃


产品详情


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.colicells. 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.
   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. colistrains using a colE1 origin of replication. Transposons made with the pMOD™-3<R6Kγori/MCS> vector also have an R6Kγoriwithin 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. colistrains 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.

 

Table 1. EZ-Tn5 pMOD Vectors
EZ-Tn5™ Transposon Construction Vectors ori that is located on vector outside of the ME sequences ori that is located within the ME sequences
pMOD™-2<MCS> colE1 None
pMOD™-3<R6Kγori/MCS> colE1 R6Kγori
pMOD™-4<MCS> R6Kγori None
pMOD™-5<R6Kγori/MCS> None R6Kγori
pMOD™-5<R6Kγori/MCS> colE1 None
*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to EPICENTRE.

 

  Catalog No. Size

EZ-Tn5™ pMOD™-2<MCS> Transposon Construction Vector
   MOD0602 20 µg

EZ-Tn5™ pMOD™3<R6Kγori/MCS> Transposon Construction Vector
   MOD1503 20 µg

EZ-Tn5™ pMOD™-4<MCS> Transposon Construction Vector
   MOD4804 20 µg

EZ-Tn5™ pMOD™-5<R6Kγori/MCS> Transposon Construction Vector
   MOD4805 20 µg

EZ-Tn5™ pMOD™-6<KAN-2/MCS> Transposon Construction Vector
   MOD7906 20 µg

pMOD™<MCS> Forward Sequencing Primer
   MODFSP201 1 nmole

pMOD™<MCS> Reverse Sequencing Primer
   MODRSP202 1 nmole

pMOD™<MCS> Forward PCR Primer
   MODFP931 1 nmole

pMOD™<MCS> Reverse PCR Primer
   MODRP941 1 nmole