pSpot8

Yeast 2-micron plasmid for expression of fusion proteins with a C-terminal Spot-Tag®.

Sequence Author: ChromoTek

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HomePlasmidsYeast PlasmidspSpot8
EcoRI (1) SphI (9270) PacI (9203) Bsu36I (8854) AatII (8491) ZraI (8489) FseI (7800) BspQI - SapI (7508) PspFI (7091) BseYI (7087) AlwNI (6982) AhdI (6503) MscI * (5399) BclI * (5357) NdeI (5323) NcoI (8) BamHI (14) AvrII (19) PspOMI (26) ApaI (30) HindIII (32) EcoRV (41) PaeR7I - PspXI - XhoI (47) TspMI - XmaI (54) SmaI (56) NheI (61) BmtI (65) Eco53kI (70) SacI (72) stop M13 fwd DraIII (638) KasI (1127) NarI (1128) SfoI (1129) PluTI (1131) BstEII (1602) BspDI - ClaI (1720) AflII (1883) BfuAI - BspMI (2562) BsrGI (2689) Bpu10I (3479) StuI (3503) BlpI (3537) NruI (3573) PstI (3669) BstAPI (3899) pSpot8 9481 bp
EcoRI  (1)
1 site		 G A A T T C C T T A A G
SphI  (9270)
1 site		 G C A T G C C G T A C G
PacI  (9203)
1 site		 T T A A T T A A A A T T A A T T
Bsu36I  (8854)
1 site		 C C T N A G G G G A N T C C
Sticky ends from different Bsu36I sites may not be compatible.
AatII  (8491)
1 site		 G A C G T C C T G C A G
ZraI  (8489)
1 site		 G A C G T C C T G C A G
FseI  (7800)
1 site		 G G C C G G C C C C G G C C G G
FseI gradually loses activity when stored at -20°C.
BspQI  (7508)
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  (7508)
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.
PspFI  (7091)
1 site		 C C C A G C G G G T C G
BseYI  (7087)
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.
AlwNI  (6982)
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.
AhdI  (6503)
1 site		 G A C N N N N N G T C C T G N N N N N C A G
The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
MscI  (5399)
1 site		 T G G C C A A C C G G T
* Blocked by Dcm methylation.
BclI  (5357)
1 site		 T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
NdeI  (5323)
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.
NcoI  (8)
1 site		 C C A T G G G G T A C C
BamHI  (14)
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.
AvrII  (19)
1 site		 C C T A G G G G A T C C
PspOMI  (26)
1 site		 G G G C C C C C C G G G
ApaI  (30)
1 site		 G G G C C C C C C G G G
ApaI can be used between 25°C and 37°C.
HindIII  (32)
1 site		 A A G C T T T T C G A A
EcoRV  (41)
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.
PaeR7I  (47)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (47)
1 site		 V C T C G A G B B G A G C T C V
XhoI  (47)
1 site		 C T C G A G G A G C T C
TspMI  (54)
1 site		 C C C G G G G G G C C C
XmaI  (54)
1 site		 C C C G G G G G G C C C
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (56)
1 site		 C C C G G G G G G C C C
SmaI can be used at 37°C for brief incubations.
NheI  (61)
1 site		 G C T A G C C G A T C G
BmtI  (65)
1 site		 G C T A G C C G A T C G
Eco53kI  (70)
1 site		 G A G C T C C T C G A G
SacI  (72)
1 site		 G A G C T C C T C G A G
DraIII  (638)
1 site		 C A C N N N G T G G T G N N N C A C
Sticky ends from different DraIII sites may not be compatible.
KasI  (1127)
1 site		 G G C G C C C C G C G G
NarI  (1128)
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  (1129)
1 site		 G G C G C C C C G C G G
PluTI  (1131)
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.
BstEII  (1602)
1 site		 G G T N A C C C C A N T G G
Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BspDI  (1720)
1 site		 A T C G A T T A G C T A
ClaI  (1720)
1 site		 A T C G A T T A G C T A
AflII  (1883)
1 site		 C T T A A G G A A T T C
BfuAI  (2562)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (2562)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsrGI  (2689)
1 site		 T G T A C A A C A T G T
BsrGI is typically used at 37°C, but is even more active at 60°C.
Bpu10I  (3479)
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.
StuI  (3503)
1 site		 A G G C C T T C C G G A
BlpI  (3537)
1 site		 G C T N A G C C G A N T C G
Sticky ends from different BlpI sites may not be compatible.
NruI  (3573)
1 site		 T C G C G A A G C G C T
PstI  (3669)
1 site		 C T G C A G G A C G T C
BstAPI  (3899)
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.
2μ ori
3980 .. 5322  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
3980 .. 5322  =  1343 bp
yeast 2μ plasmid origin of replication
LEU2
1569 .. 2663  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required for leucine biosynthesis
yeast auxotrophic marker
LEU2
1569 .. 2663  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required for leucine biosynthesis
yeast auxotrophic marker
AmpR
5716 .. 6576  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5716 .. 5784  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5716 .. 6576  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   5785 .. 6576  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5716 .. 6576  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
6747 .. 7335  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6747 .. 7335  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
405 .. 860  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
405 .. 860  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
LEU2 promoter
1161 .. 1568  =  408 bp
LEU2 promoter
1161 .. 1568  =  408 bp
AmpR promoter
5611 .. 5715  =  105 bp
AmpR promoter
5611 .. 5715  =  105 bp
MCS
1 .. 73  =  73 bp
multiple cloning site
MCS
1 .. 73  =  73 bp
multiple cloning site
T7 promoter
223 .. 241  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
223 .. 241  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 fwd
248 .. 264  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
248 .. 264  =  17 bp
common sequencing primer, one of multiple similar variants
stop
109 .. 111  =  3 bp
stop codon
stop
109 .. 111  =  3 bp
stop codon
Spot-Tag
73 .. 108  =  36 bp
12 amino acids  =  1.4 kDa
Product: engineered Spot-Tag® peptide that binds with high affinity to a nanobody
Spot-Tag
73 .. 108  =  36 bp
12 amino acids  =  1.4 kDa
Product: engineered Spot-Tag® peptide that binds with high affinity to a nanobody
ORF:  169 .. 393  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  4015 .. 4305  =  291 bp
ORF:  96 amino acids  =  11.5 kDa
ORF:  5716 .. 6576  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1569 .. 2663  =  1095 bp
ORF:  364 amino acids  =  39.0 kDa
ORF:  2231 .. 2542  =  312 bp
ORF:  103 amino acids  =  11.2 kDa
ORF:  3761 .. 4000  =  240 bp
ORF:  79 amino acids  =  9.8 kDa
ORF:  4781 .. 5101  =  321 bp
ORF:  106 amino acids  =  12.9 kDa
ORF:  7760 .. 8356  =  597 bp
ORF:  198 amino acids  =  21.9 kDa
ORF:  5143 .. 5484  =  342 bp
ORF:  113 amino acids  =  13.0 kDa
ORF:  6180 .. 6446  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  9102 .. 9335  =  234 bp
ORF:  77 amino acids  =  8.6 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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