pRS424

Yeast episomal vector with a TRP1 marker and an MCS derived from pBLUESCRIPT II.
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XcmI (5496) BseRI (5483) BmgBI (5285) BbsI (5127) NsiI (4495) ScaI (3767) NmeAIII (3435) BsaI (3348) AhdI (3287) AlwNI (2810) PfoI (46) BspEI * (332) PmlI (427) BstZ17I (434) BstAPI (546) Bsu36I (709) BsgI (880) BfuAI - BspMI - PaqCI (900) PsiI (1337) DraIII (1465) BtgZI (1466) NgoMIV (1566) NaeI (1568) Acc65I (1894) KpnI (1898) PspOMI (1900) ApaI (1904) AbsI - PaeR7I - PspXI - XhoI (1909) SalI (1915) HincII (1917) BspDI - ClaI (1925) EcoRI (1942) PstI (1952) TspMI - XmaI (1954) SmaI (1956) BamHI (1960) SpeI (1966) EagI - NotI (1979) BtgI (1988) AleI (1990) SacII (1991) Eco53kI (1998) SacI (2000) lac operator BspQI - SapI (2278) AflIII - PciI (2394) pRS424 5616 bp
XcmI  (5496)
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.
BseRI  (5483)
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.
BmgBI  (5285)
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.
BbsI  (5127)
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.
NsiI  (4495)
1 site
A T G C A T T A C G T A
ScaI  (3767)
1 site
A G T A C T T C A T G A
NmeAIII  (3435)
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).
BsaI  (3348)
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.
AhdI  (3287)
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.
AlwNI  (2810)
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.
PfoI  (46)
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.
BspEI  (332)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PmlI  (427)
1 site
C A C G T G G T G C A C
BstZ17I  (434)
1 site
G T A T A C C A T A T G
BstAPI  (546)
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.
Bsu36I  (709)
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.
BsgI  (880)
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).
BfuAI  (900)
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  (900)
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.
PaqCI  (900)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PsiI  (1337)
1 site
T T A T A A A A T A T T
DraIII  (1465)
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.
BtgZI  (1466)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
NgoMIV  (1566)
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  (1568)
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.
Acc65I  (1894)
1 site
G G T A C C C C A T G G
KpnI  (1898)
1 site
G G T A C C C C A T G G
PspOMI  (1900)
1 site
G G G C C C C C C G G G
ApaI  (1904)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AbsI  (1909)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (1909)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1909)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1909)
1 site
C T C G A G G A G C T C
SalI  (1915)
1 site
G T C G A C C A G C T G
HincII  (1917)
1 site
G T Y R A C C A R Y T G
BspDI  (1925)
1 site
A T C G A T T A G C T A
ClaI  (1925)
1 site
A T C G A T T A G C T A
EcoRI  (1942)
1 site
G A A T T C C T T A A G
PstI  (1952)
1 site
C T G C A G G A C G T C
TspMI  (1954)
1 site
C C C G G G G G G C C C
XmaI  (1954)
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  (1956)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (1960)
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.
SpeI  (1966)
1 site
A C T A G T T G A T C A
EagI  (1979)
1 site
C G G C C G G C C G G C
NotI  (1979)
1 site
G C G G C C G C C G C C G G C G
BtgI  (1988)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
AleI  (1990)
1 site
C A C N N N N G T G G T G N N N N C A C
SacII  (1991)
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.
Eco53kI  (1998)
1 site
G A G C T C C T C G A G
SacI  (2000)
1 site
G A G C T C C T C G A G
BspQI  (2278)
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  (2278)
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.
AflIII  (2394)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2394)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
2μ ori
4206 .. 5548  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
4206 .. 5548  =  1343 bp
yeast 2μ plasmid origin of replication
AmpR
3214 .. 4074  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3214 .. 4005  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3214 .. 4074  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4006 .. 4074  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3214 .. 4074  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TRP1
468 .. 1142  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
468 .. 1142  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2455 .. 3043  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2455 .. 3043  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
lacZα
1479 .. 2057  =  579 bp
192 amino acids  =  20.6 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1479 .. 2057  =  579 bp
192 amino acids  =  20.6 kDa
Product: LacZα fragment of β-galactosidase
TRP1 promoter
187 .. 467  =  281 bp
TRP1 promoter
187 .. 467  =  281 bp
AmpR promoter
4075 .. 4179  =  105 bp
AmpR promoter
4075 .. 4179  =  105 bp
lac promoter
2101 .. 2131  =  31 bp
3 segments
   Segment 3:  -10  
   2101 .. 2107  =  7 bp
promoter for the E. coli lac operon
lac promoter
2101 .. 2131  =  31 bp
3 segments
   Segment 2:  
   2108 .. 2125  =  18 bp
promoter for the E. coli lac operon
lac promoter
2101 .. 2131  =  31 bp
3 segments
   Segment 1:  -35  
   2126 .. 2131  =  6 bp
promoter for the E. coli lac operon
lac promoter
2101 .. 2131  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
2077 .. 2093  =  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
2077 .. 2093  =  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).
f1 ori
1241 .. 1696  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1241 .. 1696  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
MCS
1894 .. 2001  =  108 bp
pBluescript multiple cloning site
MCS
1894 .. 2001  =  108 bp
pBluescript multiple cloning site
T7 promoter
1867 .. 1885  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1867 .. 1885  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T3 promoter
2014 .. 2032  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
2014 .. 2032  =  19 bp
promoter for bacteriophage T3 RNA polymerase
M13 fwd
1841 .. 1857  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
1841 .. 1857  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
2053 .. 2069  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
2053 .. 2069  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  718 .. 999  =  282 bp
ORF:  93 amino acids  =  10.8 kDa
ORF:  3344 .. 3610  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  4241 .. 4531  =  291 bp
ORF:  96 amino acids  =  11.5 kDa
ORF:  468 .. 1142  =  675 bp
ORF:  224 amino acids  =  24.1 kDa
ORF:  3214 .. 4074  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1479 .. 2057  =  579 bp
ORF:  192 amino acids  =  20.6 kDa
ORF:  5007 .. 5327  =  321 bp
ORF:  106 amino acids  =  12.9 kDa
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