From Tol2Kit
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=== Construction details === | === Construction details === | ||
| − | p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction. Sequencing confirms that all bases of the insert are correct. | + | p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction with pDONR P4-P1R. Sequencing confirms that all bases of the insert are correct. |
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| + | M13F, M13R, and T3 should all work as sequencing primers. | ||
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| + | Unfortunately, the backbone of P4-P1R contains recognition sites for several potentially useful restriction enzymes. The restriction sites from the Bluescript MCS that remain unique in p5E-MCS are: | ||
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| + | ''Asp718-KpnI-DraII-XhoI-SalI-Bsp106-HindIII-SmaI-BamHI-SpeI-SacII-BstXI'' | ||
=== Crudely annotated sequence === | === Crudely annotated sequence === | ||
Revision as of 02:22, 29 September 2007
Construction details
p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction with pDONR P4-P1R. Sequencing confirms that all bases of the insert are correct.
M13F, M13R, and T3 should all work as sequencing primers.
Unfortunately, the backbone of P4-P1R contains recognition sites for several potentially useful restriction enzymes. The restriction sites from the Bluescript MCS that remain unique in p5E-MCS are:
Asp718-KpnI-DraII-XhoI-SalI-Bsp106-HindIII-SmaI-BamHI-SpeI-SacII-BstXI
Crudely annotated sequence
FASTA file with the full-length sequence as well as sequences of individual components:
p5E-MCS sequence
Crude map
Screenshot from Sequencher showing locations of components:
