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pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that all bases of the insert are correct. | pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that all bases of the insert are correct. | ||
| − | + | The following sites from the Bluescript MCS remain unique in pME-MCS: | |
| + | |||
| + | ''KpnI-XhoI-SalI-Bsp106-HindIII-EcoRI-PstI-SmaI-BamHI-SpeI-XbaI-NotI-XmaIII-SacII-BstXI-SacI'' | ||
| + | |||
| + | === Sequencing primers === | ||
| + | The insert contains the entire M13F sequence, and the 3' end of the M13R sequence. Therefore M13F will bind both in the vector backbone and in the insert, and M13R will bind perfectly to the vector backbone and weakly in the insert. There are also T7 sites in both the insert and the vector backbone. | ||
| − | The | + | The T3 site at one end of the insert should be unique and work for sequencing. |
| − | + | Clearly we did not think carefully about sequencing primers when designing this clone. Sorry. | |
=== Crudely annotated sequence === | === Crudely annotated sequence === | ||
Revision as of 02:14, 29 September 2007
Construction details
pME-MCS was made by PCR of the pBSII SK+ multiple cloning site, from M13F to M13R primers, followed by a BP reaction with pDONR221. Sequencing confirms that all bases of the insert are correct.
The following sites from the Bluescript MCS remain unique in pME-MCS:
KpnI-XhoI-SalI-Bsp106-HindIII-EcoRI-PstI-SmaI-BamHI-SpeI-XbaI-NotI-XmaIII-SacII-BstXI-SacI
Sequencing primers
The insert contains the entire M13F sequence, and the 3' end of the M13R sequence. Therefore M13F will bind both in the vector backbone and in the insert, and M13R will bind perfectly to the vector backbone and weakly in the insert. There are also T7 sites in both the insert and the vector backbone.
The T3 site at one end of the insert should be unique and work for sequencing.
Clearly we did not think carefully about sequencing primers when designing this clone. Sorry.
Crudely annotated sequence
FASTA file with the full-length sequence as well as sequences of individual components:
pME-MCS sequence
Crude map
Screenshot from Sequencher showing locations of components:
