PHO80cds

From 2007.igem.org

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'''Gene Pho80
+
GenePho
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CODING SEQUENCE'''
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CODING SEQUENCE
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ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG
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1    ATGGAAAGCA CATCAGGAGA ACGTTCCGAA AATATACATG AGGATCAAGG
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AGGATCAAGGGATACCAAAAGTAATTCTGCCCGCTGATTT
 +
TAATAAATGCTCTAGAACTGACCTAGTGGTGCTCATATCA
 +
CGAATGTTAGTATCGCTGATAGCAATCAATGAAAATTCAG
 +
CAACAAAGAAATCTGATGACCAAATTACTTTAACACGATA
 +
CCATTCTAAGATTCCTCCAAACATATCAATCTTCAACTAT
 +
TTCATACGACTGACAAAGTTTTCCTCTTTAGAACATTGTG
 +
TGCTTATGACATCACTCTATTATATCGATTTATTGCAAAC
 +
TGTGTATCCTGATTTTACGCTTAATTCGTTGACTGCCCAT
 +
AGGTTTTTATTAACAGCCACCACAGTCGCAACAAAAGGCT
 +
TATGTGATTCGTTCTCAACAAACGCCCATTATGCAAAAGT
 +
TGGAGGAGTACGATGTCACGAATTGAATATACTGGAGAAC
 +
GATTTTTTAAAGAGAGTAAACTACAGAATCATTCCGCGGG
 +
ATCATAACATTACGTTATGTAGTATAGAGCAAAAACAGAA
 +
AAAGTTTGTCATAGATAAAAACGCATTAGGGTCTCTCGAT
 +
TTGGATTCTTATTCTTACGTTAATCGTCCAAAAAGTGGAT
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ATAATGTTCTAGATAAATACTATCGAAGAATAGTTCAGCT
 +
GGTGGGTTCCTTTAACGCTTCACCTGATAAGAGTAGAAAG
 +
GTTGATTATGTTCTCCCGCCAAATATTGATATAGTGAGTG
 +
AAAGTGGCTCCCAAACTACTCAACTAAAGGGGTCGTCATC
 +
ACCCAATTCTCACTCTTCACAAAAGCGATATTCTGAGGCA
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AAGGACGCACATATCTATAACAAGCGATCAAAGCCAGATT
 +
AA
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51  GATACCAAAA GTAATTCTGC CCGCTGATTT TAATAAATGC TCTAGAACT
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XbaI restriction sequence TCTAGA
 +
cuts in sites
 +
91
 +
648
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101  ACCTAGTGGT GCTCATATCA CGAATGTTAG TATCGCTGAT AGCAATCAAT
 
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151  GAAAATTCAG CAACAAAGAA ATCTGATGAC CAAATTACTT TAACACGATA
 
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201  CCATTCTAAG ATTCCTCCAA ACATATCAAT CTTCAACTAT TTCATACGAC
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Pho cds
 +
882bp 293aa
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251  TGACAAAGTT TTCCTCTTTA GAACATTGTG TGCTTATGAC ATCACTCTAT
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1)91 bp position 31-32aa SR
 +
S=TCTTCCTCATCG
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301  TATATCGATT TATTGCAAAC TGTGTATCCT GATTTTACGC TTAATTCGTT
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R=AGAAGG
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351  GACTGCCCAT AGGTTTTTAT TAACAGCCAC CACAGTCGCA ACAAAAGGCT
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2)648 bp position 216-217-218aa VLD
 +
V=TTGTCGTAGTG
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401  TATGTGATTC GTTCTCAACA AACGCCCATT ATGCAAAAGT TGGAGGAGTA
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L=TTATTGCTTCTACTCCTG
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451  CGATGTCACG AATTGAATAT ACTGGAGAAC GATTTTTTAA AGAGAGTAAA
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D=GATGAC
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501  CTACAGAATC ATTCCGCGGG ATCATAACAT TACGTTATGT AGTATAGAGC
 
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551  AAAAACAGAA AAAGTTTGTC ATAGATAAAA ACGCATTAGG GTCTCTCGAT
 
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601  TTGGATTCTT ATTCTTACGT TAATCGTCCA AAAAGTGGAT ATAATGTTCT
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Frame 5’-3’
 +
VirtualSequence:
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651  AGATAAATAC TATCGAAGAA TAGTTCAGCT GGTGGGTTCC TTTAACGCTT
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MESTSGERSENIHEDQGIPKVILPADFNKC[[SR]]TDLVVLISRMLVSLIAINENSATKKSDD
 +
QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH
 +
RFLLTATTVATKGLCDSFSTNAHYAKVGGVRCHELNILENDFLKRVNYRIIPRDHNITLC
 +
SIEQKQKKFVIDKNALGSLDLDSYSYVNRPKSGYN[[VLD]]KYYRRIVQLVGSFNASPDKSRK
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VDYVLPPNIDIVSESGSQTTQLKGSSSPNSHSSQKRYSEAKDAHIYNKRSKPD
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701  CACCTGATAA GAGTAGAAAG GTTGATTATG TTCTCCCGCC AAATATTGAT
 
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751  ATAGTGAGTG AAAGTGGCTC CCAAACTACT CAACTAAAGG GGTCGTCATC
 
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801  ACCCAATTCT CACTCTTCAC AAAAGCGATA TTCTGAGGCA AAGGACGCAC
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'''PHO80
 +
Primer sequences:'''
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851  ATATCTATAA CAAGCGATCA AAGCCAGATT AA
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t93c            5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'
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t93c antisense  5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'
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XbaI cuts in 2 sites 91 and 648
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t648c          5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'
 +
t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'
 +
'''
 +
results of pho80 coding sequence'''
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'''Pho80cds'''
 
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882 bp e 293aa
 
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XbaI restriction sequence TCTAGA
 
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1)91 bp position 31-32aa SR
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''PCR of mutagenesis of pho 80 from topo TA plasmid''
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S=TCT  TCC  TCA  TCG
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Finally volume is 50μl
 +
{| border="1"
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|-
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! Acqua, µl
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| 40
 +
|-
 +
! Buffer, µl
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| 5
 +
|-
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! dNTPs, µl
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| 1
 +
|-
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! Taq (Pfu turbo), µl
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| 1
 +
|-
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! PrimerFw (125 ng), µl
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| 1
 +
|-
 +
! PrimerRev (125 ng), µl
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| 1
 +
|-
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! Template (5 ng), µl
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| 1
 +
|}
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R=AGA  AGG
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Cycling Conditions
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{| border="1"
 +
|-
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! I°CYCLE
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| 95°c for 30 seconds
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|-
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! 12 CYCLES
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| denaturation  95°C for 30 seconds
 +
| annealing    55°c for 1 minutes
 +
| elongation    68°c for 5 minutes
 +
|-
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! LAST CYCLE
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| 72°C for 2 minutes
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| 4°c
 +
|}
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2)648 bp position 216-217-218aa VLD
 
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V=TT    GTC    GTA    GTG
 
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L=TTA  TTG    CTT    CTA  CTC  CTG
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PRIMERS
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D=GAT  GAC
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PHO 80 fw Xba I
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5'- GC TCTAGA ATGGAAAGCACATCAGGAG-3'
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Tm 65.1°C
 +
PHO 80 rew Spe I
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5'-GG ACTAGT ATCTGGCTTTGATCGCTTG-3'
 +
Tm 65.1°C
 +
[[PCR]] of pho80 from DNA yeast
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5'3' Frame 1
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[[Image:geldoc pho 1.jpg]]
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Met E S T S G E R S E N I H E D Q G I P K V I L P A D F N K C S R T D L V V L I S R Met L V S L I A I N E N S A T K K S D D Q I T L T R Y H S K I P P N I S I F N Y F I R L T K F S S L E H C V L Met T S L Y Y I D L L Q T V Y P D F T L N S L T A H R F L L T A T T V A T K G L C D S F S T N A H Y A K V G G V R C H E L N I L E N D F L K R V N Y R I I P R D H N I T L C S I E Q K Q K K F V I D K N A L G S L D L D S Y S Y V N R P K S G Y N V L D K Y Y R R I V Q L V G S F N A S P D K S R K V D Y V L P P N I D I V S E S G S Q T T Q L K G S S S P N S H S S Q K R Y S E A K D A H I Y N K R S K P D Stop
 
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Virtual Sequence:
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midi pho80 in topo TA plasmid
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SEQUENCE  293 AA;  33227 MW; 72918CAF845AE74C CRC64.
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    MESTSGERSE NIHEDQGIPK VILPADFNKC [[SR]]TDLVVLIS RMLVSLIAIN ENSATKKSDD
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    QITLTRYHSK IPPNISIFNY FIRLTKFSSL EHCVLMTSLY YIDLLQTVYP DFTLNSLTAH
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    RFLLTATTVA TKGLCDSFST NAHYAKVGGV RCHELNILEN DFLKRVNYRI IPRDHNITLC
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    SIEQKQKKFV IDKNALGSLD LDSYSYVNRP KSGYN[[VLD]]KY YRRIVQLVGS FNASPDKSRK
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    VDYVLPPNID IVSESGSQTT QLKGSSSPNS HSSQKRYSEA KDAHIYNKRS KPD
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 +
[[Image:geldoc pho 2.jpg]]
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'''PHO80
 
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Primer sequences:'''
 
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t93c            5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'
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digestion with Dpn I after first mutation of pho 80
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t93c antisense  5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'
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[[Image:geldoc pho 3.jpg]]
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t648c          5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'
 
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t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'
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digestion with Dpn I after second mutation of pho 80
 +
 
 +
[[Image:geldoc pho 4.jpg]]
 +
 
 +
 
 +
midi pho 80 mutated
 +
 
 +
[[Image:geldoc pho 5.jpg]]
 +
 
 +
 
 +
digestion pho 80 mutated with Spe i and Xba I
 +
 
 +
[[Image:geldoc pho 6.jpg]]

Latest revision as of 16:53, 25 October 2007

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

Geldoc pho 1.jpg


midi pho80 in topo TA plasmid

Geldoc pho 2.jpg


digestion with Dpn I after first mutation of pho 80

Geldoc pho 3.jpg


digestion with Dpn I after second mutation of pho 80

Geldoc pho 4.jpg


midi pho 80 mutated

Geldoc pho 5.jpg


digestion pho 80 mutated with Spe i and Xba I

Geldoc pho 6.jpg

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