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Plasmid Files

YEplac195

Yeast episomal plasmid with a URA3 marker.

 
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 DrdI (5013) AhdI (4227) BsrFI (4142) NmeAIII (4080) TsoI (3829) AatII (3308) ZraI (3306) NdeI (3087) BsgI (3058) PpuMI (2846) EcoRV (2814) NcoI (2793) BstBI (2735) BsmI (2642) lac operator HindIII (233) BfuAI - BspMI (238) SphI (243) PstI - SbfI (249) SalI (251) XbaI (257) BamHI (263) TspMI - XmaI (268) SmaI (270) Acc65I (272) KpnI (276) Eco53kI (280) SacI (282) EcoRI (284) KasI (445) NarI (446) SfoI (447) PluTI (449) PfoI * (640) BstAPI (662) HpaI (742) BsaAI - SnaBI (1384) BseRI (2024) MfeI (2038) BspDI * - ClaI * (2088) PsiI (2187) Bpu10I (2269) BsmBI (2376) StuI (2566) PspOMI * (2624) ApaI * (2628) YEplac195 5241 bp
DrdI  (5013)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
AhdI  (4227)
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.
BsrFI  (4142)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
NmeAIII  (4080)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
TsoI  (3829)
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).
AatII  (3308)
1 site
G A C G T C C T G C A G
ZraI  (3306)
1 site
G A C G T C C T G C A G
NdeI  (3087)
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.
BsgI  (3058)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PpuMI  (2846)
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.
EcoRV  (2814)
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.
NcoI  (2793)
1 site
C C A T G G G G T A C C
BstBI  (2735)
1 site
T T C G A A A A G C T T
BsmI  (2642)
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.
HindIII  (233)
1 site
A A G C T T T T C G A A
BfuAI  (238)
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  (238)
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.
SphI  (243)
1 site
G C A T G C C G T A C G
PstI  (249)
1 site
C T G C A G G A C G T C
SbfI  (249)
1 site
C C T G C A G G G G A C G T C C
SalI  (251)
1 site
G T C G A C C A G C T G
XbaI  (257)
1 site
T C T A G A A G A T C T
BamHI  (263)
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.
TspMI  (268)
1 site
C C C G G G G G G C C C
XmaI  (268)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (270)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Acc65I  (272)
1 site
G G T A C C C C A T G G
KpnI  (276)
1 site
G G T A C C C C A T G G
Eco53kI  (280)
1 site
G A G C T C C T C G A G
SacI  (282)
1 site
G A G C T C C T C G A G
EcoRI  (284)
1 site
G A A T T C C T T A A G
KasI  (445)
1 site
G G C G C C C C G C G G
NarI  (446)
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  (447)
1 site
G G C G C C C C G C G G
PluTI  (449)
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.
PfoI  (640)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BstAPI  (662)
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.
HpaI  (742)
1 site
G T T A A C C A A T T G
BsaAI  (1384)
1 site
Y A C G T R R T G C A Y
SnaBI  (1384)
1 site
T A C G T A A T G C A T
BseRI  (2024)
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.
MfeI  (2038)
1 site
C A A T T G G T T A A C
BspDI  (2088)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (2088)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PsiI  (2187)
1 site
T T A T A A A A T A T T
Bpu10I  (2269)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
BsmBI  (2376)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
StuI  (2566)
1 site
A G G C C T T C C G G A
PspOMI  (2624)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI  (2628)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI can be used between 25°C and 37°C.
2μ ori
745 .. 2091  =  1347 bp
yeast 2μ plasmid origin of replication
2μ ori
745 .. 2091  =  1347 bp
yeast 2μ plasmid origin of replication
AmpR
3440 .. 4300  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3440 .. 3508  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3440 .. 4300  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3509 .. 4300  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3440 .. 4300  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
URA3
2200 .. 3003  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase,
required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)
URA3
2200 .. 3003  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase,
required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)
ori
4471 .. 5059  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
4471 .. 5059  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
lacZα
216 .. 500  =  285 bp
95 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
lacZα
216 .. 500  =  285 bp
95 amino acids  =  11.0 kDa
Product: LacZα fragment of β-galactosidase
URA3 promoter
3004 .. 3224  =  221 bp
URA3 promoter
3004 .. 3224  =  221 bp
AmpR promoter
3335 .. 3439  =  105 bp
AmpR promoter
3335 .. 3439  =  105 bp
lac promoter
142 .. 172  =  31 bp
   Segment 1:  -35  
   142 .. 147  =  6 bp
promoter for the E. coli lac operon
lac promoter
142 .. 172  =  31 bp
   Segment 2:  
   148 .. 165  =  18 bp
promoter for the E. coli lac operon
lac promoter
142 .. 172  =  31 bp
   Segment 3:  -10  
   166 .. 172  =  7 bp
promoter for the E. coli lac operon
lac promoter
142 .. 172  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
180 .. 196  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
180 .. 196  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
MCS
233 .. 289  =  57 bp
pUC19 multiple cloning site
MCS
233 .. 289  =  57 bp
pUC19 multiple cloning site
M13 rev
204 .. 220  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
204 .. 220  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
290 .. 306  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
290 .. 306  =  17 bp
common sequencing primer, one of multiple similar
variants
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