CFP (kan) [plasmid pDH3]

Plamid pDH3 contains the ORF for the cyan-fluorescent protein (CFP), a variant of GFP with a blue-shifted spectrum. The plasmid also contains the Kan selectable marker encoding resistance to the antibiotic G418. The codons have not been optimized for mammalian expression and instead are the same as in the parent vector (except for the 6 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.

Map

Parent Vector

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

Mutations

Amino Acid Change Original Codon New Codon
F64L TTC TTG
Y66W TAT TGG
N146I AAC ATT
M153T ATG ACT
V163A GTT GCT
N164H AAT CAT
Also includes S65T and Q80R from parent vector. Q80R is believed to be a neutral mutation.

Sequence

The DNA sequence of pDH3 is shown below with the CFP sequence in blue.
GAACGCGGCCGCCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCCC GGGTTAATTAACAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAAT TCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATT TGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTT GACTTGGGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGC ATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACT ATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTT TGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTA AAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATATTTCA CACAATGTATACATCACTGCAGACAAACAAAAGAATGGAATCAAAGCTCA TTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACC ATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC AACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAA GAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACAC ATGGCATGGATGAACTATACAAATAGGGCGCGCCACTTCTAAATAAGCGA ATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATA AGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTT ATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGT ATGAGGTCGCTCTTATTGACCACACCTCTACCGGCAGATCCGCTAGGGAT AACAGGGTAATATAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACC CGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTG CGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACA TCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGC GAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCT CCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAA TATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTT TTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAA CCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAAC ATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCA ATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGT TGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAG ATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAA GCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCC CCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAA AATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCC TGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGG CGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGAC GAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCT TTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTG ATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGA CGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTG CCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATG GTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGAT GAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAAC TTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAA TGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATG CGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATG CTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTTTA AACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGG CCGCGGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGC CAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGG TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGG TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGG CCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTA TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT TTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA TTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGAT ACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGG GCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATG ATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCA AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT AAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT CTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCA CGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG GCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT ATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATG TATCATACACATACGATTTAGGTGACACTATA

Plasmids & Protocols