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Plasmid Files

pRS

HuSH-29 retroviral vector with ampicillin and puromycin resistance markers, for expressing an shRNA from the U6 promoter.

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pRS Sequence and MappRS.dna
Map and Sequence File   
Sequence Author:  OriGene
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 NaeI (5420) NgoMIV (5418) BsrGI (5300) Bsu36I (4807) SpeI (4686) AarI - BfuAI - BspMI (4078) SspI (3959) XmnI (3754) PvuI (3525) FspI (3377) NotI (3034) EcoRI (1) DraIII - PmlI (40) NdeI (268) BamHI (334) HindIII (380) SalI (386) AccI (387) SphI (464) NsiI (466) NcoI (551) SfiI (597) AvrII (644) BsiWI (729) BspEI * (786) RsrII (789) SacII (887) BspDI - ClaI (1307) BspQI - SapI (2139) AflIII - PciI (2255) DrdI (2363) pRS 5432 bp
NaeI  (5420)
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.
NgoMIV  (5418)
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.
BsrGI  (5300)
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.
Bsu36I  (4807)
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.
SpeI  (4686)
1 site
A C T A G T T G A T C A
AarI  (4078)
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 AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BfuAI  (4078)
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  (4078)
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.
SspI  (3959)
1 site
A A T A T T T T A T A A
XmnI  (3754)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuI  (3525)
1 site
C G A T C G G C T A G C
FspI  (3377)
1 site
T G C G C A A C G C G T
NotI  (3034)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1)
1 site
G A A T T C C T T A A G
DraIII  (40)
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.
PmlI  (40)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NdeI  (268)
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.
BamHI  (334)
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.
HindIII  (380)
1 site
A A G C T T T T C G A A
SalI  (386)
1 site
G T C G A C C A G C T G
AccI  (387)
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.
SphI  (464)
1 site
G C A T G C C G T A C G
NsiI  (466)
1 site
A T G C A T T A C G T A
NcoI  (551)
1 site
C C A T G G G G T A C C
SfiI  (597)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
AvrII  (644)
1 site
C C T A G G G G A T C C
BsiWI  (729)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BspEI  (786)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
RsrII  (789)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
SacII  (887)
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.
BspDI  (1307)
1 site
A T C G A T T A G C T A
ClaI  (1307)
1 site
A T C G A T T A G C T A
BspQI  (2139)
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  (2139)
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  (2255)
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  (2255)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (2363)
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.
AmpR
3082 .. 3942  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3082 .. 3873  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3082 .. 3942  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   3874 .. 3942  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3082 .. 3942  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
673 .. 1272  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
673 .. 1272  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
3' LTR
1351 .. 1944  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
3' LTR
1351 .. 1944  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
ori
2316 .. 2904  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2316 .. 2904  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
5' LTR
4078 .. 4547  =  470 bp
long terminal repeat from Moloney murine leukemia
virus
5' LTR
4078 .. 4547  =  470 bp
long terminal repeat from Moloney murine leukemia
virus
gag (truncated)
5004 .. 5420  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
5004 .. 5420  =  417 bp
truncated MMLV gag gene lacking the start codon
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
MMLV Ψ
4612 .. 4811  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
MMLV Ψ
4612 .. 4811  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
SV40 promoter
463 .. 659  =  197 bp
SV40 early promoter
SV40 promoter
463 .. 659  =  197 bp
SV40 early promoter
AmpR promoter
3943 .. 4047  =  105 bp
AmpR promoter
3943 .. 4047  =  105 bp
shRNA site
334 .. 385  =  52 bp
shRNA insertion site
shRNA site
334 .. 385  =  52 bp
shRNA insertion site
SV40 ori
510 .. 645  =  136 bp
SV40 origin of replication
SV40 ori
510 .. 645  =  136 bp
SV40 origin of replication
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