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Plasmid Files

pMSCVhyg

Retroviral vector with a hygromycin resistance marker optimized for gene expression in hematopoietic, embryonic stem, or embryonic carcinoma cells.

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pMSCVhyg Sequence and MappMSCVhyg.dna
Map and Sequence File   
Sequence Author:  Clontech
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 SgrAI (6767) PfoI (6257) AflIII - PciI (4331) BspQI - SapI (4215) BspDI - ClaI (3293) BstEII (1088) SexAI * (1216) BsrGI (1283) 5' pMSCV primer (1333 .. 1355) BglII (1410) PaeR7I - XhoI (1416) HpaI (1424) AgeI (1548) 3' pMSCV primer (1660 .. 1682) StuI (1732) BsmI (1869) PshAI (1982) NcoI (2307) AsiSI (2319) RsrII (2363) BtgZI (2627) SacII (2735) AleI (3206) SalI (3272) AccI (3273) HindIII (3286) pMSCVhyg 6945 bp
SgrAI  (6767)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
PfoI  (6257)
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.
AflIII  (4331)
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  (4331)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4215)
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  (4215)
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.
BspDI  (3293)
1 site
A T C G A T T A G C T A
ClaI  (3293)
1 site
A T C G A T T A G C T A
BstEII  (1088)
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.
SexAI  (1216)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BsrGI  (1283)
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.
BglII  (1410)
1 site
A G A T C T T C T A G A
PaeR7I  (1416)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1416)
1 site
C T C G A G G A G C T C
HpaI  (1424)
1 site
G T T A A C C A A T T G
AgeI  (1548)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
StuI  (1732)
1 site
A G G C C T T C C G G A
BsmI  (1869)
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.
PshAI  (1982)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
NcoI  (2307)
1 site
C C A T G G G G T A C C
AsiSI  (2319)
1 site
G C G A T C G C C G C T A G C G
RsrII  (2363)
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.
BtgZI  (2627)
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.
SacII  (2735)
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.
AleI  (3206)
1 site
C A C N N N N G T G G T G N N N N C A C
SalI  (3272)
1 site
G T C G A C C A G C T G
AccI  (3273)
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.
HindIII  (3286)
1 site
A A G C T T T T C G A A
5' pMSCV primer
23-mer  /  61% GC
1 binding site
1333 .. 1355  =  23 annealed bases
Tm  =  62°C
3' pMSCV primer
23-mer  /  48% GC
1 binding site
1660 .. 1682  =  23 annealed bases
Tm  =  58°C
HygR
1955 .. 2980  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
1955 .. 2980  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
AmpR
5151 .. 6011  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5151 .. 5942  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5151 .. 6011  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5943 .. 6011  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5151 .. 6011  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4392 .. 4980  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4392 .. 4980  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
5' LTR
6944 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
5' LTR
6944 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
3' LTR
3338 .. 3852  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
3' LTR
3338 .. 3852  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
PGK promoter
1438 .. 1937  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
1438 .. 1937  =  500 bp
mouse phosphoglycerate kinase 1 promoter
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell
virus
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell
virus
AmpR promoter
6012 .. 6116  =  105 bp
AmpR promoter
6012 .. 6116  =  105 bp
MCS
1410 .. 1433  =  24 bp
multiple cloning site
MCS
1410 .. 1433  =  24 bp
multiple cloning site
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