YFP (his3) [plasmid pDH5]

Plasmid pDH5 contains the ORF for a pH-tolerant yellow-fluorescent protein (YFP), a variant of GFP. The plasmid also contains the selectable marker from S. pombe that complements S. cerevisiae his3. The codons have not been optimized for mammalian expression; instead the codons are the same as in the parent vector (except for the 5 mutations listed below). Forward and reverse primers refer to the sequence of the primers described on the protocol page "Fusing GFP to the Carboxy Terminus of Your Favorite Protein." Some common restriction enzyme sites are shown. Unique sites are in red. His5 is from S. pombe and complements His3 in S. cerevisiae.

Map

Parent Vector

pFA6a-GFP(S65T)-HIS. See Wach et al.

Mutations in the GFP(S65T) ORF

Amino acid Change Original Codon New Codon
S65G ACT GGT
V68L GTT CTA
Q69K CAA AAA
S72A TCA GCT
T203Y ACA TAT
Also includes Q80R from parent vector. Q80R is believed to be a neutral mutation.

Sequence

The DNA sequence of pDH5 with the YFP sequence in green.
GAACGCGGCCGCCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCC CGGGTTAATTAACAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCA ATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCA GTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATT TATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACT ACTTTCGGTTATGGTCTAAAATGCTTTGCTAGATACCCAGATCATATGA AACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGA AAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAA GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA TTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAA CTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGA ATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTC AACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGT CCTTTTACCAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAA GATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAG CTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGGGCGCGCC ACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAAT AAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGT TTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGG TTGCTTTCTCAGGTATAGTATGAGGTCGCTCTTATTGACCACACCTCTA CCGGCAGATCCGCTAGGGATAACAGGGTAATATAGATCTGTTTAGCTTG CCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATAC CCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGT CGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCA TACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCT GCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGT CCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCAC TGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAG TTCTCACATCACATCCGAACATAAACAACCATGGGTAGGAGGGCTTTTG TAGAAAGAAATACGAACGAAACGAAAATCAGCGTTGCCATCGCTTTGGA CAAAGCTCCCTTACCTGAAGAGTCGAATTTTATTGATGAACTTATAACT TCCAAGCATGCAAACCAAAAGGGAGAACAAGTAATCCAAGTAGACACGG GAATTGGATTCTTGGATCACATGTATCATGCACTGGCTAAACATGCAGG CTGGAGCTTACGACTTTACTCAAGAGGTGATTTAATCATCGATGATCAT CACACTGCAGAAGATACTGCTATTGCACTTGGTATTGCATTCAAGCAGG CTATGGGTAACTTTGCCGGCGTTAAAAGATTTGGACATGCTTATTGTCC ACTTGACGAAGCTCTTTCTAGAAGCGTAGTTGACTTGTCGGGACGGCCC TATGCTGTTATCGATTTGGGATTAAAGCGTGAAAAGGTTGGGGAATTGT CCTGTGAAATGATCCCTCACTTACTATATTCCTTTTCGGTAGCAGCTGG AATTACTTTGCATGTTACCTGCTTATATGGTAGTAATGACCATCATCGT GCTGAAAGCGCTTTTAAATCTCTGGCTGTTGCCATGCGCGCGGCTACTA GTCTTACTGGAAGTTCTGAAGTCCCAAGCACGAAGGGAGTGTTGTAAAG AGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTAT AGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGAT TTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGT GCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTA TACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTTTAAACGAGCTC GAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGGCCGCGGAT CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTA ACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTAT CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCA GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTG TGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGAT CCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGT GTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCA ATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGT TCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCG TGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATAC GGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGG AAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT TTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGC CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATT ATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCC GGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCC CGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACT ATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATATT GTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACAC ATACGATTTAGGTGACACTATA

Plasmids & Protocols