PDestTol2pA*: Difference between revisions
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== Construction Details == | == Construction Details == | ||
pDestTol2pA was built by Clemens Grabher. The R4-R3 cassette including the attR4 recombination site, ccdB gene, chloramphenicol resistance gene and the attR3 recombination site was amplified by PCR from the original pDESTR4-R3 vector (Invitrogen) using the following primers: | |||
<pre> | |||
M13 REV/XHOI: 5’-ACCGGTCTCGAGCAGGAAACAGCTATGAC-3’ | |||
and | |||
M13 FWD/CLAI/KPNI: 5’-AGCCGTATCGATGGTACCGTAAAACGACGGCCAG-3’. | |||
</pre> | |||
The PCR product was subcloned into pCRII using the TOPO TA cloning kit (Invitrogen) and verified by sequencing. Subsequently, the insert was subcloned into the Tol2 vector pT2KXIGDin (K. Kawakami) using ClaI/XhoI, resulting in the Tol2 destination vector pDestTol2pA | |||
== Crudely Annotated Sequence == | |||
FASTA file with the full-length sequence as well as sequences of individual components): pDestTol2pA sequence | |||
Edited 7 June 2007 to add six bases (a KpnI site that was missing). | |||
== Crude Map == | == Crude Map == | ||
Screenshot from Sequencher showing locations of components: | Screenshot from Sequencher showing locations of components: | ||
PDestTol2pA.png | |||
Revision as of 16:56, 13 March 2025
Construction Details
pDestTol2pA was built by Clemens Grabher. The R4-R3 cassette including the attR4 recombination site, ccdB gene, chloramphenicol resistance gene and the attR3 recombination site was amplified by PCR from the original pDESTR4-R3 vector (Invitrogen) using the following primers:
M13 REV/XHOI: 5’-ACCGGTCTCGAGCAGGAAACAGCTATGAC-3’ and M13 FWD/CLAI/KPNI: 5’-AGCCGTATCGATGGTACCGTAAAACGACGGCCAG-3’.
The PCR product was subcloned into pCRII using the TOPO TA cloning kit (Invitrogen) and verified by sequencing. Subsequently, the insert was subcloned into the Tol2 vector pT2KXIGDin (K. Kawakami) using ClaI/XhoI, resulting in the Tol2 destination vector pDestTol2pA
Crudely Annotated Sequence
FASTA file with the full-length sequence as well as sequences of individual components): pDestTol2pA sequence
Edited 7 June 2007 to add six bases (a KpnI site that was missing).
Crude Map
Screenshot from Sequencher showing locations of components: PDestTol2pA.png