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YIplac211

Yeast integrating plasmid with a URA3 marker.

 
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YIplac211.dna
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HindIII (233) AhdI (2783) BsrFI (2698) NmeAIII (2636) TsoI (2385) XmnI (2183) SspI (1978) AatII (1864) BfuAI - BspMI (238) SphI (243) PstI - SbfI (249) SalI (251) XbaI (257) BamHI (263) AvaI - BsoBI - TspMI - XmaI (268) BmeT110I (269) SmaI (270) Acc65I (272) KpnI (276) Eco53kI (280) SacI (282) EcoRI (284) KasI (445) NarI (446) SfoI (447) PluTI (449) BstAPI (502) PfoI (633) NsiI (753) PsiI (761) Bpu10I (843) StuI (1140) PspOMI * (1198) ApaI * (1202) BsmI (1216) BbsI (1298) BstBI (1309) NcoI (1367) EcoRV (1388) EcoO109I - PpuMI (1420) XcmI (1449) BsgI (1632) ZraI (1862) YIplac211 3797 bp
HindIII  (233)
1 site
A A G C T T T T C G A A
AhdI  (2783)
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  (2698)
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  (2636)
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  (2385)
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).
XmnI  (2183)
1 site
G A A N N N N T T C C T T N N N N A A G
SspI  (1978)
1 site
A A T A T T T T A T A A
AatII  (1864)
1 site
G A C G T C C T G C A G
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.
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
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.
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.
NsiI  (753)
1 site
A T G C A T T A C G T A
PsiI  (761)
1 site
T T A T A A A A T A T T
Bpu10I  (843)
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.
StuI  (1140)
1 site
A G G C C T T C C G G A
PspOMI  (1198)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI  (1202)
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.
BsmI  (1216)
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.
BbsI  (1298)
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.
BstBI  (1309)
1 site
T T C G A A A A G C T T
NcoI  (1367)
1 site
C C A T G G G G T A C C
EcoRV  (1388)
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.
EcoO109I  (1420)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
PpuMI  (1420)
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.
XcmI  (1449)
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.
BsgI  (1632)
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).
ZraI  (1862)
1 site
G A C G T C C T G C A G
AmpR
1996 .. 2856  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1996 .. 2064  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1996 .. 2856  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2065 .. 2856  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1996 .. 2856  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
URA3
774 .. 1577  =  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
774 .. 1577  =  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
3027 .. 3615  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
3027 .. 3615  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
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
URA3 promoter
1578 .. 1798  =  221 bp
URA3 promoter
1578 .. 1798  =  221 bp
AmpR promoter
1891 .. 1995  =  105 bp
AmpR promoter
1891 .. 1995  =  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:  1066 .. 1542  =  477 bp
ORF:  158 amino acids  =  16.8 kDa
ORF:  1996 .. 2856  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  216 .. 539  =  324 bp
ORF:  107 amino acids  =  12.2 kDa
ORF:  774 .. 1577  =  804 bp
ORF:  267 amino acids  =  29.3 kDa
ORF:  2460 .. 2726  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  143 .. 499  =  357 bp
ORF:  118 amino acids  =  13.5 kDa
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