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Plasmid Files

pMSCVpuro

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

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pMSCVpuro.dna
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Sequence Author:  Clontech (TaKaRa)
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SgrAI (6086) NdeI (5714) ScaI (5023) AflIII - PciI (3650) BspQI - SapI (3534) BsrGI (1283) 5' pMSCV primer (1333 .. 1355) BglII (1410) PaeR7I - XhoI (1416) HincII - HpaI (1424) EcoRI (1428) AgeI (1548) 3' pMSCV primer (1660 .. 1682) BlpI (1711) BsmI (1869) BfuAI - BspMI (1947) HindIII (1949) BsiWI (2014) RsrII (2074) SacII (2172) DraIII (2529) NsiI (2611) BspDI - ClaI (2612) pMSCVpuro 6264 bp
SgrAI  (6086)
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.
NdeI  (5714)
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.
ScaI  (5023)
1 site
A G T A C T T C A T G A
AflIII  (3650)
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  (3650)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3534)
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  (3534)
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.
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
HincII  (1424)
1 site
G T Y R A C C A R Y T G
HpaI  (1424)
1 site
G T T A A C C A A T T G
EcoRI  (1428)
1 site
G A A T T C C T T A A G
AgeI  (1548)
1 site
A C C G G T T G G C C A
BlpI  (1711)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
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.
BfuAI  (1947)
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  (1947)
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.
HindIII  (1949)
1 site
A A G C T T T T C G A A
BsiWI  (2014)
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.
RsrII  (2074)
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  (2172)
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.
DraIII  (2529)
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.
NsiI  (2611)
1 site
A T G C A T T A C G T A
BspDI  (2612)
1 site
A T C G A T T A G C T A
ClaI  (2612)
1 site
A T C G A T T A G C T 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
AmpR
4470 .. 5330  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4470 .. 5261  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4470 .. 5330  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5262 .. 5330  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4470 .. 5330  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
PuroR
1958 .. 2557  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
1958 .. 2557  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
3711 .. 4299  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3711 .. 4299  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
5' LTR
6263 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
5' LTR
6263 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
3' LTR
2657 .. 3171  =  515 bp
3' long terminal repeat from murine embryonic stem cell virus
3' LTR
2657 .. 3171  =  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
5331 .. 5435  =  105 bp
AmpR promoter
5331 .. 5435  =  105 bp
MCS
1410 .. 1433  =  24 bp
multiple cloning site
MCS
1410 .. 1433  =  24 bp
multiple cloning site
ORF:  4600 .. 4866  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5716 .. 6012  =  297 bp
ORF:  98 amino acids  =  11.7 kDa
ORF:  1718 .. 2557  =  840 bp
ORF:  279 amino acids  =  30.1 kDa
ORF:  4470 .. 5330  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  362 .. 637  =  276 bp
ORF:  91 amino acids  =  9.9 kDa
ORF:  674 .. 934  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  1883 .. 2608  =  726 bp
ORF:  241 amino acids  =  24.8 kDa
ORF:  5780 .. 6010  =  231 bp
ORF:  76 amino acids  =  7.5 kDa
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