(New page: === 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 al...) |
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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 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. | |
| − | + | === Sequence === | |
| + | Annotated sequence, Genbank format:<br> | ||
| + | [[pME-MCS Genbank]] | ||
| − | |||
FASTA file with the full-length sequence as well as sequences of individual components:<br> | FASTA file with the full-length sequence as well as sequences of individual components:<br> | ||
[[pME-MCS sequence]] | [[pME-MCS sequence]] | ||
| + | |||
| + | Note that there are late stop codons in two of the reading frames. So, if you are trying to make a "no stop" middle clone, you must pick the correct frame. Note that you will also add a C-terminal fusion of at least 16 amino acids to your protein. It may be better to do a BP reaction with pDONR 221 if you want to make a "no stop" clone. | ||
=== Crude map === | === Crude map === | ||
Screenshot from Sequencher showing locations of components:<br> | Screenshot from Sequencher showing locations of components:<br> | ||
[[Image:pME-MCS.png]] | [[Image:pME-MCS.png]] | ||
Latest revision as of 19:19, 22 January 2010
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.
Sequence
Annotated sequence, Genbank format:
pME-MCS Genbank
FASTA file with the full-length sequence as well as sequences of individual components:
pME-MCS sequence
Note that there are late stop codons in two of the reading frames. So, if you are trying to make a "no stop" middle clone, you must pick the correct frame. Note that you will also add a C-terminal fusion of at least 16 amino acids to your protein. It may be better to do a BP reaction with pDONR 221 if you want to make a "no stop" clone.
Crude map
Screenshot from Sequencher showing locations of components:
