The construct GFP-KDEL was made from a part of the ECast/PDI gene and includes the S65T GFP mutant of Heim, Cubitt and Tsien.
From the ECast/PDI mRNA sequence, we used the seven nucleotide untranslated kozak sequence (cttaaaa), 17 amino acid signal sequence and first 11 amino acids of the mature form of ECast/PDI. There was a two amino acid linker (leucine glutamic) followed by the entire S65T GFP mutant. At the end was the 4 amino acid ER retention sequence KDEL.
GFP-KDEL is in the vector pSP64-S, with EcoR1 (start) and Bgl2 (end) restriction sites. Use BamH1 for linearizing the vector. mRNA was made using the RNA polymerase SP6; the mRNA made contains 5' and 3' untranslated regions of xenopus globin mRNA .
Nucleotide sequence (from EcoR1 to Bgl 2 sites)
gaattccttaaaaatgaagtatttggctctttgttttattgctctggcct
gtgcggtccatgccgcagtcgaggtcgaaatcgaagaagatgtcgctctc
gagatgagtaaaggagaagaacttttcactggagttgtcccaattcttgt
tgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagg
gtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcact
actggaaaactacctgttccatggccaacacttgtcactactttctctta
tggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgact
ttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattt
ttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaagg
tgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaag
atggaaacattcttggacacaaattggaatacaactataactcacacaat
gtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaa
aattagacacaacattgaagatggaagcgttcaactagcagaccattatc
aacaaaatactccaattggcgatggccctgtccttttaccagacaaccat
tacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagaga
ccacatggtccttcttgagtttgtaacagctgctgggattacacatggca
tggatgaactatacaaaaaggatgaactttaaagatct
Krieg, P.A., and Melton, D.A. 1984. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Research 12: 7057-7070.
The globin 5' and 3' untranslated regions are from Xenopus.
Annotated nucleotide sequence of SP64-S in the vicinity of the SP6 promoter
Lucero, H.A., Lebeche, D., and Kaminer, B. 1994. ER calcistorin / protein disulfide isomerase (PDI): sequence determination and expression of cDNA clone encoding a calcium storage protein with PDI activity from endoplasmic reticulum of the sea urchin egg. J. Biol. Chem. 269: 23112-23119.
Heim, R., Cubitt, A.B., and Tsien, R.Y. 1995. Improved green fluorescence. Nature 373: 663-664.
Munro, S., and Pelham, H.R. 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899-907.
