(New page: === Construction details === p3E-EGFPpA was made by PCR amplification of EGFP and an SV40 late polyadenylation signal from a pCS2+-based plasmid, using primers to add att sites, followed b...) |
|||
| (2 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
p3E-EGFPpA was made by PCR amplification of EGFP and an SV40 late polyadenylation signal from a pCS2+-based plasmid, using primers to add att sites, followed by a BP reaction. | p3E-EGFPpA was made by PCR amplification of EGFP and an SV40 late polyadenylation signal from a pCS2+-based plasmid, using primers to add att sites, followed by a BP reaction. | ||
| − | Note that the EGFP sequence is followed by 60 bp of polylinker sequence from pCS2+ before the stop codon. We have confirmed functionally that this cassette produces fluorescent protein. Sequencing confirms that all bases of the insert are correct. | + | Note that the EGFP sequence is followed by 60 bp of polylinker sequence from pCS2+ before the stop codon. We have confirmed functionally that this cassette produces fluorescent protein in C-terminal fusion constructs. Sequencing confirms that all bases of the insert are correct. |
| + | |||
| + | The EGFP sequence '''does''' include a start codon and a good Kozak consensus, so it is possible to use this clone to test promoters cloned into a middle clone. | ||
| + | |||
| + | === Sequence === | ||
| + | Annotated sequence, Genbank format:<br> | ||
| + | [[p3E-EGFPpA 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> | ||
[[p3E-EGFPpA sequence]] | [[p3E-EGFPpA sequence]] | ||
Latest revision as of 03:52, 19 December 2007
Construction details
p3E-EGFPpA was made by PCR amplification of EGFP and an SV40 late polyadenylation signal from a pCS2+-based plasmid, using primers to add att sites, followed by a BP reaction.
Note that the EGFP sequence is followed by 60 bp of polylinker sequence from pCS2+ before the stop codon. We have confirmed functionally that this cassette produces fluorescent protein in C-terminal fusion constructs. Sequencing confirms that all bases of the insert are correct.
The EGFP sequence does include a start codon and a good Kozak consensus, so it is possible to use this clone to test promoters cloned into a middle clone.
Sequence
Annotated sequence, Genbank format:
p3E-EGFPpA Genbank
FASTA file with the full-length sequence as well as sequences of individual components:
p3E-EGFPpA sequence
Crude map
Screenshot from Sequencher showing locations of components:
