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Plasmid Files

YIplac128

Yeast integrating plasmid with a LEU2 marker.

 
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YIplac128.dna
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BspQI - SapI (4284) PspFI (3867) BseYI (3863) AlwNI (3758) AhdI (3279) BsaI (3213) NmeAIII (3132) ScaI (2798) AatII (2360) ZraI (2358) HindIII (233) SphI (243) PstI - SbfI (249) SalI (251) AccI (252) XbaI (257) BamHI (263) AvaI - BsoBI - TspMI - XmaI (268) BmeT110I (269) SmaI (270) Acc65I (272) KpnI (276) Eco53kI (280) BanII - SacI (282) EcoRI (284) KasI (445) NarI (446) SfoI (447) PluTI (449) NdeI (498) BstAPI (502) PfoI (633) BsrGI (766) EcoRV (1141) BstXI (1180) XcmI (1335) BbsI (1435) AflII (1572) BspDI - ClaI (1737) BstEII (1852) PflMI (1983) PpuMI (1999) SacII (2089) YIplac128 4293 bp
BspQI  (4284)
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  (4284)
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  (3867)
1 site
C C C A G C G G G T C G
BseYI  (3863)
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  (3758)
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  (3279)
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.
BsaI  (3213)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
NmeAIII  (3132)
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).
ScaI  (2798)
1 site
A G T A C T T C A T G A
AatII  (2360)
1 site
G A C G T C C T G C A G
ZraI  (2358)
1 site
G A C G T C C T G C A G
HindIII  (233)
1 site
A A G C T T T T C G A A
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
AccI  (252)
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.
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.
AvaI  (268)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (268)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
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.
BmeT110I  (269)
1 site
C Y C G R G G R G C Y C
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
BanII  (282)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
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.
NdeI  (498)
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.
BstAPI  (502)
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.
PfoI  (633)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
BsrGI  (766)
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.
EcoRV  (1141)
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.
BstXI  (1180)
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.
XcmI  (1335)
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.
BbsI  (1435)
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.
AflII  (1572)
1 site
C T T A A G G A A T T C
BspDI  (1737)
1 site
A T C G A T T A G C T A
ClaI  (1737)
1 site
A T C G A T T A G C T A
BstEII  (1852)
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.
PflMI  (1983)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
PpuMI  (1999)
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.
SacII  (2089)
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.
LEU2
797 .. 1891  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required for leucine biosynthesis
yeast auxotrophic marker
LEU2
797 .. 1891  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required for leucine biosynthesis
yeast auxotrophic marker
AmpR
2492 .. 3352  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2492 .. 2560  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2492 .. 3352  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2561 .. 3352  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2492 .. 3352  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3523 .. 4111  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
3523 .. 4111  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
LEU2 promoter
1892 .. 2299  =  408 bp
LEU2 promoter
1892 .. 2299  =  408 bp
lacZα
216 .. 539  =  324 bp
107 amino acids  =  12.2 kDa
Product: LacZα fragment of β-galactosidase
lacZα
216 .. 539  =  324 bp
107 amino acids  =  12.2 kDa
Product: LacZα fragment of β-galactosidase
AmpR promoter
2387 .. 2491  =  105 bp
AmpR promoter
2387 .. 2491  =  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
ORF:  2492 .. 3352  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  216 .. 539  =  324 bp
ORF:  107 amino acids  =  12.2 kDa
ORF:  918 .. 1229  =  312 bp
ORF:  103 amino acids  =  11.1 kDa
ORF:  511 .. 753  =  243 bp
ORF:  80 amino acids  =  9.2 kDa
ORF:  2956 .. 3222  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  143 .. 499  =  357 bp
ORF:  118 amino acids  =  13.5 kDa
ORF:  797 .. 1891  =  1095 bp
ORF:  364 amino acids  =  39.0 kDa
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