pAG25

Plasmid for yeast gene deletion using the natMX4 selectable marker conferring nourseothricin resistance. See also pAG29 and pAG32.
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HomePlasmidsYeast PlasmidspAG25
PvuII (15) NdeI (3621) AatII (3372) ZraI (3370) SspI (3254) XmnI (3049) TsoI (2849) PvuI (2820) BmrI (2490) AlwNI (1973) PspFI (1865) HindIII (19) SalI (37) AccI (38) BamHI (43) PacI (58) AscI (63) BglII (70) BstXI (117) BmgBI (153) Bpu10I (162) BseRI (264) NcoI (457) AgeI (487) XcmI (530) PpuMI (541) BbsI (554) PflFI - Tth111I (558) PshAI (702) NgoMIV (769) NaeI (771) NruI (846) KasI (870) NarI (871) SfoI (872) PluTI (874) SgrAI (911) SphI (1019) BstAPI (1021) BsmI (1221) Eco53kI (1278) SacI (1280) ApoI - EcoRI (1282) BspDI - ClaI (1289) EcoRV (1296) SpeI (1306) SfiI (1319) SacII (1326) HpaI (1378) BspQI - SapI (1441) PciI (1557) BseYI (1861) pAG25 3704 bp
PvuII  (15)
1 site		 C A G C T G G T C G A C
NdeI  (3621)
1 site		 C A T A T G G T A T A C
Prolonged incubation with NdeI may lead to removal of additional nucleotides.
AatII  (3372)
1 site		 G A C G T C C T G C A G
ZraI  (3370)
1 site		 G A C G T C C T G C A G
SspI  (3254)
1 site		 A A T A T T T T A T A A
XmnI  (3049)
1 site		 G A A N N N N T T C C T T N N N N A A G
TsoI  (2849)
1 site		 T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9
Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuI  (2820)
1 site		 C G A T C G G C T A G C
BmrI  (2490)
1 site		 A C T G G G ( N ) 4 N T G A C C C ( N ) 4
The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
AlwNI  (1973)
1 site		 C A G N N N C T G G T C N N N G A C
Sticky ends from different AlwNI sites may not be compatible.
PspFI  (1865)
1 site		 C C C A G C G G G T C G
HindIII  (19)
1 site		 A A G C T T T T C G A A
SalI  (37)
1 site		 G T C G A C C A G C T G
AccI  (38)
1 site		 G T M K A C C A K M T G
Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BamHI  (43)
1 site		 G G A T C C C C T A G G
After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
PacI  (58)
1 site		 T T A A T T A A A A T T A A T T
AscI  (63)
1 site		 G G C G C G C C C C G C G C G G
BglII  (70)
1 site		 A G A T C T T C T A G A
BstXI  (117)
1 site		 C C A N N N N N N T G G G G T N N N N N N A C C
Sticky ends from different BstXI sites may not be compatible.
BmgBI  (153)
1 site		 C A C G T C G T G C A G
This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
Bpu10I  (162)
1 site		 C C T N A G C G G A N T C G
Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BseRI  (264)
1 site		 G A G G A G ( N ) 8 N N C T C C T C ( N ) 8
Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
NcoI  (457)
1 site		 C C A T G G G G T A C C
AgeI  (487)
1 site		 A C C G G T T G G C C A
XcmI  (530)
1 site		 C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C
The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
PpuMI  (541)
1 site		 R G G W C C Y Y C C W G G R
Sticky ends from different PpuMI sites may not be compatible.
BbsI  (554)
1 site		 G A A G A C N N C T T C T G N N ( N ) 4
Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
PflFI  (558)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (558)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PshAI  (702)
1 site		 G A C N N N N G T C C T G N N N N C A G
PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
NgoMIV  (769)
1 site		 G C C G G C C G G C C G
Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (771)
1 site		 G C C G G C C G G C C G
Efficient cleavage requires at least two copies of the NaeI recognition sequence.
NruI  (846)
1 site		 T C G C G A A G C G C T
KasI  (870)
1 site		 G G C G C C C C G C G G
NarI  (871)
1 site		 G G C G C C C C G C G G
Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (872)
1 site		 G G C G C C C C G C G G
PluTI  (874)
1 site		 G G C G C C C C G C G G
Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SgrAI  (911)
1 site		 C R C C G G Y G G Y G G C C R C
Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
SphI  (1019)
1 site		 G C A T G C C G T A C G
BstAPI  (1021)
1 site		 G C A N N N N N T G C C G T N N N N N A C G
Sticky ends from different BstAPI sites may not be compatible.
BsmI  (1221)
1 site		 G A A T G C N C T T A C G N
Sticky ends from different BsmI sites may not be compatible.
Eco53kI  (1278)
1 site		 G A G C T C C T C G A G
SacI  (1280)
1 site		 G A G C T C C T C G A G
ApoI  (1282)
1 site		 R A A T T Y Y T T A A R
ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (1282)
1 site		 G A A T T C C T T A A G
BspDI  (1289)
1 site		 A T C G A T T A G C T A
ClaI  (1289)
1 site		 A T C G A T T A G C T A
EcoRV  (1296)
1 site		 G A T A T C C T A T A G
EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
SpeI  (1306)
1 site		 A C T A G T T G A T C A
SfiI  (1319)
1 site		 G G C C N N N N N G G C C C C G G N N N N N C C G G
Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
SacII  (1326)
1 site		 C C G C G G G G C G C C
Efficient cleavage requires at least two copies of the SacII recognition sequence.
HpaI  (1378)
1 site		 G T T A A C C A A T T G
BspQI  (1441)
1 site		 G C T C T T C N C G A G A A G N N N N
Sticky ends from different BspQI sites may not be compatible.
SapI  (1441)
1 site		 G C T C T T C N C G A G A A G N N N N
Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
PciI  (1557)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BseYI  (1861)
1 site		 C C C A G C G G G T C G
After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
natMX6
115 .. 1234  =  1120 bp
yeast selectable marker conferring nourseothricin resistance
natMX6
115 .. 1234  =  1120 bp
yeast selectable marker conferring nourseothricin resistance
AmpR
2377 .. 3237  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2377 .. 3168  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2377 .. 3237  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3169 .. 3237  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2377 .. 3237  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1618 .. 2206  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1618 .. 2206  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3238 .. 3342  =  105 bp
AmpR promoter
3238 .. 3342  =  105 bp
T7 promoter
1342 .. 1360  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1342 .. 1360  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
3688 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
3688 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
NrsR
459 .. 1031  =  573 bp
190 amino acids  =  20.5 kDa
Product: nourseothricin acetyltransferase
confers resistance to nourseothricin
NrsR
459 .. 1031  =  573 bp
190 amino acids  =  20.5 kDa
Product: nourseothricin acetyltransferase
confers resistance to nourseothricin
TEF promoter
115 .. 458  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
115 .. 458  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
1037 .. 1234  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1037 .. 1234  =  198 bp
Ashbya gossypii TEF terminator
ORF:  459 .. 1031  =  573 bp
ORF:  190 amino acids  =  20.5 kDa
ORF:  2377 .. 3237  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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Individual Sequences & Maps

bleMX6
pFA6a-kanMX6
pPIC6(alpha) B
pYES2.1-E
hphMX6
pFA6a-kanMX6-PGAL1
pPIC6(alpha) C
pYES2 CT
kanMX
pFA6a-kanMX6-PGAL1-3HA
pPIC9
pYES2 NT A
kanMX2
pFA6a-kanMX6-PGAL1-GFP
pPIC9K
pYES2 NT B
kanMX4
pFA6a-kanMX6-PGAL1-GST
pPICZ A
pYES2 NT C
kanMX6
pFA6a-natMX6
pPICZ B
pYES3 CT
natMX6
pFA6a-TRP1
pPICZ C
pYES6 CT
pAbAi
pFA6a-TRP1-PGAL1
pPICZ(alpha) A
pYM25
pAD-GAL4
pFA6a-TRP1-PGAL1-3HA
pPICZ(alpha) B
pYM26
pAG25
pFA6a-TRP1-PGAL1-GFP
pPICZ(alpha) C
pYM44
pAG29
pFA6a-TRP1-PGAL1-GST
pPink-HC
YCp50
pAG32
pFLD
pPink-LC
YCp50-poly
pAO815
pFLD(alpha)
pPINK(alpha)-HC
YCplac111
patMX4
pGAD-C1
pRS303
YCplac22
pAUR101
pGAD-C2
pRS304
YCplac33
pAUR112
pGAD-C3
pRS305
YEp13
pAUR123
pGADT7 AD
pRS306
YEp24
pAUR224
pGADT7-Rec
pRS313
YEp351
pBridge
pGADT7-Rec (linearized)
pRS314
YEp352
pDH25
pGAPZ A
pRS315
YEp353
pEG202
pGAPZ B
pRS316
YEp354
pESC-HIS
pGAPZ C
pRS403
YEp355
pESC-LEU
pGAPZ(alpha) A
pRS404
YEp356
pESC-TRP
pGAPZ(alpha) B
pRS405
YEp356R
pESC-URA
pGAPZ(alpha) C
pRS406
YEp357
pFA6-kanMX4
pGBD-C1
pRS413
YEp357R
pFA6a
pGBD-C2
pRS414
YEp358
pFA6a-12Pk-kanMX6
pGBD-C3
pRS415
YEp358R
pFA6a-13Myc-His3MX6
pGBDU-C1
pRS416
YEp363
pFA6a-13Myc-kanMX6
pGBDU-C2
pRS423
YEp364
pFA6a-13Myc-TRP1
pGBDU-C3
pRS424
YEp365
pFA6a-3HA-His3MX6
pGBKT7
pRS425
YEp366
pFA6a-3HA-kanMX6
pGilda
pRS426
YEp366R
pFA6a-3HA-TRP1
pHIL-D2
pSpot5
YEp367
pFA6a-5FLAG-kanMX6
pHIL-S1
pSpot6
YEp367R
pFA6a-bleMX6
pJG4-5
pSpot7
YEp368
pFA6a-GFP(S65T)-His3MX6
pKLAC1
pSpot8
YEp368R
pFA6a-GFP(S65T)-kanMX6
pKLAC2
pTEF1 Zeo
YEplac112
pFA6a-GFP(S65T)-TRP1
pOAD
pUCATPH
YEplac181
pFA6a-GST-His3MX6
pOBD2
pY-secSUMOstar
YEplac195
pFA6a-GST-kanMX6
pP-secSUMO3
pY-SUMOstar
YIp351
pFA6a-GST-TRP1
pPIC3
pYC2-E
YIp352
pFA6a-His3MX6
pPIC3.5
pYC2 CT
YIp5
pFA6a-His3MX6-PGAL1
pPIC3.5K
pYC2 NT A
YIplac128
pFA6a-His3MX6-PGAL1-3HA
pPIC6 A
pYC2 NT B
YIplac204
pFA6a-His3MX6-PGAL1-GFP
pPIC6 B
pYC2 NT C
YIplac211
pFA6a-His3MX6-PGAL1-GST
pPIC6 C
pYC6 CT
YRp7
pFA6a-hphMX6
pPIC6(alpha) A
pYES2
alpha-factor secretion signal

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