From 2007.igem.org

Revision as of 15:41, 16 October 2007 (view source) Alda (Talk | contribs) ← Older edit		Latest revision as of 16:53, 25 October 2007 (view source) Giov1984 (Talk | contribs)
(39 intermediate revisions not shown)
Line 1:		Line 1:
-	Gene Pho80	+	GenePho
	CODING SEQUENCE		CODING SEQUENCE
-	1 ATGGAAAGCA CATCAGGAGA ACGTTCCGAA AATATACATG AGGATCAAGG	+	ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATG
		+	AGGATCAAGGGATACCAAAAGTAATTCTGCCCGCTGATTT
		+	TAATAAATGCTCTAGAACTGACCTAGTGGTGCTCATATCA
		+	CGAATGTTAGTATCGCTGATAGCAATCAATGAAAATTCAG
		+	CAACAAAGAAATCTGATGACCAAATTACTTTAACACGATA
		+	CCATTCTAAGATTCCTCCAAACATATCAATCTTCAACTAT
		+	TTCATACGACTGACAAAGTTTTCCTCTTTAGAACATTGTG
		+	TGCTTATGACATCACTCTATTATATCGATTTATTGCAAAC
		+	TGTGTATCCTGATTTTACGCTTAATTCGTTGACTGCCCAT
		+	AGGTTTTTATTAACAGCCACCACAGTCGCAACAAAAGGCT
		+	TATGTGATTCGTTCTCAACAAACGCCCATTATGCAAAAGT
		+	TGGAGGAGTACGATGTCACGAATTGAATATACTGGAGAAC
		+	GATTTTTTAAAGAGAGTAAACTACAGAATCATTCCGCGGG
		+	ATCATAACATTACGTTATGTAGTATAGAGCAAAAACAGAA
		+	AAAGTTTGTCATAGATAAAAACGCATTAGGGTCTCTCGAT
		+	TTGGATTCTTATTCTTACGTTAATCGTCCAAAAAGTGGAT
		+	ATAATGTTCTAGATAAATACTATCGAAGAATAGTTCAGCT
		+	GGTGGGTTCCTTTAACGCTTCACCTGATAAGAGTAGAAAG
		+	GTTGATTATGTTCTCCCGCCAAATATTGATATAGTGAGTG
		+	AAAGTGGCTCCCAAACTACTCAACTAAAGGGGTCGTCATC
		+	ACCCAATTCTCACTCTTCACAAAAGCGATATTCTGAGGCA
		+	AAGGACGCACATATCTATAACAAGCGATCAAAGCCAGATT
		+	AA
-	51  GATACCAAAA GTAATTCTGC CCGCTGATTT TAATAAATGC TCTAGAACTG	+	XbaI restriction sequence TCTAGA
		+	cuts in sites
		+	91
		+	648
-	101  ACCTAGTGGT GCTCATATCA CGAATGTTAG TATCGCTGAT AGCAATCAAT
-	151  GAAAATTCAG CAACAAAGAA ATCTGATGAC CAAATTACTT TAACACGATA
-	201  CCATTCTAAG ATTCCTCCAA ACATATCAAT CTTCAACTAT TTCATACGAC	+	Pho cds
		+	882bp 293aa
-	251  TGACAAAGTT TTCCTCTTTA GAACATTGTG TGCTTATGAC ATCACTCTAT	+	1)91 bp position 31-32aa SR
		+	S=TCTTCCTCATCG
-	301  TATATCGATT TATTGCAAAC TGTGTATCCT GATTTTACGC TTAATTCGTT	+	R=AGAAGG
-	351  GACTGCCCAT AGGTTTTTAT TAACAGCCAC CACAGTCGCA ACAAAAGGCT	+	2)648 bp position 216-217-218aa VLD
		+	V=TTGTCGTAGTG
-	401  TATGTGATTC GTTCTCAACA AACGCCCATT ATGCAAAAGT TGGAGGAGTA	+	L=TTATTGCTTCTACTCCTG
-	451  CGATGTCACG AATTGAATAT ACTGGAGAAC GATTTTTTAA AGAGAGTAAA	+	D=GATGAC
-	501  CTACAGAATC ATTCCGCGGG ATCATAACAT TACGTTATGT AGTATAGAGC
-	551  AAAAACAGAA AAAGTTTGTC ATAGATAAAA ACGCATTAGG GTCTCTCGAT
-	601  TTGGATTCTT ATTCTTACGT TAATCGTCCA AAAAGTGGAT ATAATGTTCT
-	651  AGATAAATAC TATCGAAGAA TAGTTCAGCT GGTGGGTTCC TTTAACGCTT	+	Frame 5’-3’
		+	VirtualSequence:
-	701  CACCTGATAA GAGTAGAAAG GTTGATTATG TTCTCCCGCC AAATATTGAT	+	MESTSGERSENIHEDQGIPKVILPADFNKC[[SR]]TDLVVLISRMLVSLIAINENSATKKSDD
		+	QITLTRYHSKIPPNISIFNYFIRLTKFSSLEHCVLMTSLYYIDLLQTVYPDFTLNSLTAH
		+	RFLLTATTVATKGLCDSFSTNAHYAKVGGVRCHELNILENDFLKRVNYRIIPRDHNITLC
		+	SIEQKQKKFVIDKNALGSLDLDSYSYVNRPKSGYN[[VLD]]KYYRRIVQLVGSFNASPDKSRK
		+	VDYVLPPNIDIVSESGSQTTQLKGSSSPNSHSSQKRYSEAKDAHIYNKRSKPD
-	751  ATAGTGAGTG AAAGTGGCTC CCAAACTACT CAACTAAAGG GGTCGTCATC
-	801  ACCCAATTCT CACTCTTCAC AAAAGCGATA TTCTGAGGCA AAGGACGCAC
-	851  ATATCTATAA CAAGCGATCA AAGCCAGATT AA	+	'''PHO80
		+	Primer sequences:'''
-	XbaI cuts in 2 sites	+	t93c            5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'
-	•91	+	t93c antisense  5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'
-	•648	+
-		+
		+	t648c          5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'
		+	t648c antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'
-	Pho80cds	+	'''
-	882 bp e 293aa	+	results of pho80 coding sequence'''
-	XbaI restriction sequence TCTAGA
-	1)91 bp position 31-32aa SR
-	S=TCT  TCC  TCA  TCG
-	R=AGA  AGG	+	''PCR of mutagenesis of pho 80 from topo TA plasmid''
		+	Finally volume is 50μl
		+	{| border="1"
		+	|-
		+	! 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
-	2)648 bp position 216-217-218aa VLD	+	{| border="1"
-	V=TT   GTC    GTA    GTG	+	|-
		+	! 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
		+
		+	[[Image:geldoc pho 1.jpg]]
		+
		+
		+	midi pho80 in topo TA plasmid
		+
		+	[[Image:geldoc pho 2.jpg]]
		+
		+
		+	digestion with Dpn I after first mutation of pho 80
-	L=TTA  TTG    CTT    CTA  CTC  CTG	+	[[Image:geldoc pho 3.jpg]]
-	D=GAT  GAC
		+	digestion with Dpn I after second mutation of pho 80
		+	[[Image:geldoc pho 4.jpg]]
-	5'3' Frame 1
-	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
-	Virtual Sequence: VIRT14212	+	midi pho 80 mutated
-	ID  VIRT14212              Unreviewed;      293 AA.	+
-	AC  VIRT14212;	+
-	DE  Translation of nucleotide sequence generated on ExPASy	+
-	DE  on 10-Jul-2007 by 193.43.101.20.	+
-	CC  -!- This virtual protein sequence will automatically be deleted	+
-	CC      from the server after a few days.	+
-	CC  9.11 PI.	+
-	DR  SWISS-2DPAGE; VIRT14212; VIRTUAL.	+
-	SQ  SEQUENCE  293 AA;  33227 MW; 72918CAF845AE74C CRC64.	+
-	MESTSGERSE NIHEDQGIPK VILPADFNKC [[SR]]TDLVVLIS RMLVSLIAIN ENSATKKSDD	+
-	QITLTRYHSK IPPNISIFNY FIRLTKFSSL EHCVLMTSLY YIDLLQTVYP DFTLNSLTAH	+
-	RFLLTATTVA TKGLCDSFST NAHYAKVGGV RCHELNILEN DFLKRVNYRI IPRDHNITLC	+
-	SIEQKQKKFV IDKNALGSLD LDSYSYVNRP KSGYN[[VLD]]KY YRRIVQLVGS FNASPDKSRK	+
-	VDYVLPPNID IVSESGSQTT QLKGSSSPNS HSSQKRYSEA KDAHIYNKRS KPD	+
		+	[[Image:geldoc pho 5.jpg]]
-	PHO80
-	Primer sequences:
-	Primer Name Primer Sequence (5' to 3')
-	t93c 5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'
-	t93c_antisense 5'-gcaccactaggtcagttctggagcatttattaaaatcagcg-3'
-	t648c 5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'
-	t648c_antisense 5'-ctattcttcgatagtatttatctaggacattatatccactttttggacgat-3'
-	Oligonucleotide information:
-	Primer Name Length (nt.) Tm Duplex Energy at 65°C Energy Cost of Mismatches
-	t93c 41 79.60°C -44.47 kcal/mole 3.0%
-	t93c_antisense 41 79.60°C -46.01 kcal/mole 2.6%
-	t648c 51 78.36°C -46.13 kcal/mole 0.1%
-	t648c_antisense 51 78.36°C -42.85 kcal/mole 1.8%
-	Primer-template duplexes:
-	Primer Name Primer-Template Duplex
-	t93c 5'-cgctgattttaataaatgctccagaactgacctagtggtgc-3'
-	||||||||||||||||||||| |||||||||||||||||||
-	cgggcgactaaaattatttacgagatcttgactggatcaccacgagt
-	t93c_antisense gcccgctgattttaataaatgctctagaactgacctagtggtgctca
-	||||||||||||||||||||| |||||||||||||||||||
-	3'-gcgactaaaattatttacgaggtcttgactggatcaccacg-5'
-	t648c 5'-atcgtccaaaaagtggatataatgtcctagataaatactatcgaagaatag-3'
-	||||||||||||||||||||||||| |||||||||||||||||||||||||
-	aattagcaggtttttcacctatattacaagatctatttatgatagcttcttatcaag
-	t648c_antisense ttaatcgtccaaaaagtggatataatgttctagataaatactatcgaagaatagttc
-	||||||||||||||||||||||||| |||||||||||||||||||||||||
-	3'-tagcaggtttttcacctatattacaggatctatttatgatagcttcttatc-5'
		+	digestion pho 80 mutated with Spe i and Xba I
-	Primer sequences:	+	[[Image:geldoc pho 6.jpg]]
-	Primer Name Primer Sequence (5' to 3')	+
-	t93g 5'-cgctgattttaataaatgctcgagaactgacctagtggtgc-3'	+
-	t93g_antisense 5'-gcaccactaggtcagttctcgagcatttattaaaatcagcg-3'	+
-	Oligonucleotide information:	+
-	Primer Name Length (nt.) Tm Duplex Energy at 68°C Energy Cost of Mismatches	+
-	t93g 41 79.60°C -46.43 kcal/mole 1.3%	+
-	t93g_antisense 41 79.60°C -46.23 kcal/mole 2.2%	+
-	Primer-template duplexes:	+
-	Primer Name Primer-Template Duplex	+
-	t93g 5'-cgctgattttaataaatgctcgagaactgacctagtggtgc-3'	+
-	||||||||||||||||||||| |||||||||||||||||||	+
-	cgggcgactaaaattatttacgagatcttgactggatcaccacgagt	+
-	t93g_antisense gcccgctgattttaataaatgctctagaactgacctagtggtgctca	+
-	||||||||||||||||||||| |||||||||||||||||||	+
-	3'-gcgactaaaattatttacgagctcttgactggatcaccacg-5'	+

---

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

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 with Spe i and Xba I