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Plasmid Files

pRS306

Yeast integrative vector with a URA3 marker and an MCS derived from pBLUESCRIPT.

 
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 AatII (4308) ZraI (4306) XmnI (3985) AhdI (3386) PfoI (46) NdeI (324) BsgI (357) SbfI (393) BfuAI - BspMI (396) XcmI (539) PpuMI (564) NcoI (616) BstBI (676) BbsI (685) BsmI (773) StuI (847) Bpu10I (1141) NsiI (1238) BsaAI (1567) BtgZI (1568) NgoMIV (1668) NaeI (1670) Eco53kI (1995) SacI (1997) AleI (2003) SacII (2004) BstXI (2005) EagI - NotI (2010) XbaI (2017) SpeI (2023) BamHI (2029) TspMI - XmaI (2035) SmaI (2037) EcoRI (2047) HindIII (2059) BspDI - ClaI (2066) SalI (2074) AbsI - PaeR7I - PspXI - XhoI (2080) Acc65I (2095) KpnI (2099) lac operator pRS306 4373 bp
AatII  (4308)
1 site
G A C G T C C T G C A G
ZraI  (4306)
1 site
G A C G T C C T G C A G
XmnI  (3985)
1 site
G A A N N N N T T C C T T N N N N A A G
AhdI  (3386)
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.
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.
NdeI  (324)
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  (357)
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).
SbfI  (393)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (396)
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  (396)
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.
XcmI  (539)
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.
PpuMI  (564)
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.
NcoI  (616)
1 site
C C A T G G G G T A C C
BstBI  (676)
1 site
T T C G A A A A G C T T
BbsI  (685)
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.
BsmI  (773)
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.
StuI  (847)
1 site
A G G C C T T C C G G A
Bpu10I  (1141)
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.
NsiI  (1238)
1 site
A T G C A T T A C G T A
BsaAI  (1567)
1 site
Y A C G T R R T G C A Y
BtgZI  (1568)
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  (1668)
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  (1670)
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.
Eco53kI  (1995)
1 site
G A G C T C C T C G A G
SacI  (1997)
1 site
G A G C T C C T C G A G
AleI  (2003)
1 site
C A C N N N N G T G G T G N N N N C A C
SacII  (2004)
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.
BstXI  (2005)
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.
EagI  (2010)
1 site
C G G C C G G C C G G C
NotI  (2010)
1 site
G C G G C C G C C G C C G G C G
XbaI  (2017)
1 site
T C T A G A A G A T C T
SpeI  (2023)
1 site
A C T A G T T G A T C A
BamHI  (2029)
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  (2035)
1 site
C C C G G G G G G C C C
XmaI  (2035)
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  (2037)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
EcoRI  (2047)
1 site
G A A T T C C T T A A G
HindIII  (2059)
1 site
A A G C T T T T C G A A
BspDI  (2066)
1 site
A T C G A T T A G C T A
ClaI  (2066)
1 site
A T C G A T T A G C T A
SalI  (2074)
1 site
G T C G A C C A G C T G
AbsI  (2080)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2080)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2080)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2080)
1 site
C T C G A G G A G C T C
Acc65I  (2095)
1 site
G G T A C C C C A T G G
KpnI  (2099)
1 site
G G T A C C C C A T G G
AmpR
3313 .. 4173  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3313 .. 4104  =  792 bp
   263 amino acids  =  29.0 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3313 .. 4173  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4105 .. 4173  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3313 .. 4173  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
URA3
411 .. 1214  =  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
411 .. 1214  =  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
2554 .. 3142  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2554 .. 3142  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
lacZ'
1581 .. 2156  =  576 bp
191 amino acids  =  20.8 kDa
Product: fragment of β-galactosidase
lacZ'
1581 .. 2156  =  576 bp
191 amino acids  =  20.8 kDa
Product: fragment of β-galactosidase
URA3 promoter
190 .. 410  =  221 bp
URA3 promoter
190 .. 410  =  221 bp
AmpR promoter
4174 .. 4278  =  105 bp
AmpR promoter
4174 .. 4278  =  105 bp
lac promoter
2200 .. 2230  =  31 bp
   Segment 3:  -10  
   2200 .. 2206  =  7 bp
promoter for the E. coli lac operon
lac promoter
2200 .. 2230  =  31 bp
   Segment 2:  
   2207 .. 2224  =  18 bp
promoter for the E. coli lac operon
lac promoter
2200 .. 2230  =  31 bp
   Segment 1:  -35  
   2225 .. 2230  =  6 bp
promoter for the E. coli lac operon
lac promoter
2200 .. 2230  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
2176 .. 2192  =  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
2176 .. 2192  =  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
1343 .. 1798  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1343 .. 1798  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
MCS
1993 .. 2100  =  108 bp
pBluescript multiple cloning site
MCS
1993 .. 2100  =  108 bp
pBluescript multiple cloning site
T7 promoter
1966 .. 1984  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1966 .. 1984  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T3 promoter
2113 .. 2131  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
2113 .. 2131  =  19 bp
promoter for bacteriophage T3 RNA polymerase
M13 fwd
1943 .. 1959  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
1943 .. 1959  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2152 .. 2168  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2152 .. 2168  =  17 bp
common sequencing primer, one of multiple similar
variants
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