From 2007.igem.org

GenePho CODING SEQUENCE

ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG AGGATCAAGGGATACCAAAAGTAATTCTGCCCGCTGATTT TAATAAATGCTCTAGAACTGACCTAGTGGTGCTCATATCA CGAATGTTAGTATCGCTGATAGCAATCAATGAAAATTCAG CAACAAAGAAATCTGATGACCAAATTACTTTAACACGATA CCATTCTAAGATTCCTCCAAACATATCAATCTTCAACTAT TTCATACGACTGACAAAGTTTTCCTCTTTAGAACATTGTG TGCTTATGACATCACTCTATTATATCGATTTATTGCAAAC TGTGTATCCTGATTTTACGCTTAATTCGTTGACTGCCCAT AGGTTTTTATTAACAGCCACCACAGTCGCAACAAAAGGCT TATGTGATTCGTTCTCAACAAACGCCCATTATGCAAAAGT TGGAGGAGTACGATGTCACGAATTGAATATACTGGAGAAC GATTTTTTAAAGAGAGTAAACTACAGAATCATTCCGCGGG ATCATAACATTACGTTATGTAGTATAGAGCAAAAACAGAA AAAGTTTGTCATAGATAAAAACGCATTAGGGTCTCTCGAT TTGGATTCTTATTCTTACGTTAATCGTCCAAAAAGTGGAT ATAATGTTCTAGATAAATACTATCGAAGAATAGTTCAGCT GGTGGGTTCCTTTAACGCTTCACCTGATAAGAGTAGAAAG GTTGATTATGTTCTCCCGCCAAATATTGATATAGTGAGTG AAAGTGGCTCCCAAACTACTCAACTAAAGGGGTCGTCATC ACCCAATTCTCACTCTTCACAAAAGCGATATTCTGAGGCA AAGGACGCACATATCTATAACAAGCGATCAAAGCCAGATT AA

XbaI restriction sequence TCTAGA cuts in sites 91 648

Pho cds 882bp 293aa

1)91 bp position 31-32aa SR S=TCTTCCTCATCG

R=AGAAGG

2)648 bp position 216-217-218aa VLD V=TTGTCGTAGTG

L=TTATTGCTTCTACTCCTG

D=GATGAC

Frame 5’-3’ VirtualSequence:

MESTSGERSENIHEDQGIPKVILPADFNKCSRTDLVVLISRMLVSLIAINENSATKKSDD QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH RFLLTATTVATKGLCDSFSTNAHYAKVGGVRCHELNILENDFLKRVNYRIIPRDHNITLC SIEQKQKKFVIDKNALGSLDLDSYSYVNRPKSGYNVLDKYYRRIVQLVGSFNASPDKSRK VDYVLPPNIDIVSESGSQTTQLKGSSSPNSHSSQKRYSEAKDAHIYNKRSKPD

PHO80 Primer sequences:

t93c 5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'

t93c antisense 5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'

t648c 5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'

t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'

results of pho80 coding sequence

PCR of mutagenesis of pho 80 from topo TA plasmid Finally volume is 50μl

Acqua, µl	40
Buffer, µl	5
dNTPs, µl	1
Taq (Pfu turbo), µl	1
PrimerFw (125 ng), µl	1
PrimerRev (125 ng), µl	1
Template (5 ng), µl	1

Cycling Conditions

I°CYCLE	95°c for 30 seconds
12 CYCLES	denaturation 95°C for 30 seconds	annealing 55°c for 1 minutes	elongation 68°c for 5 minutes
LAST CYCLE	72°C for 2 minutes	4°c

PRIMERS

PHO 80 fw Xba I 5'- GC TCTAGA ATGGAAAGCACATCAGGAG-3' Tm 65.1°C

PHO 80 rew Spe I 5'-GG ACTAGT ATCTGGCTTTGATCGCTTG-3' Tm 65.1°C

PCR of pho80 from DNA yeast

midi pho80 in topo TA plasmid

digestion with Dpn I after first mutation of pho 80

digestion with Dpn I after second mutation of pho 80

midi pho 80 mutated

digestion pho 80 mutated whit Spe i and Xba I