pMSCVneo

Retroviral vector with a neomycin (G418) resistance marker optimized for gene expression in hematopoietic, embryonic stem, or embryonic carcinoma cells.

Sequence Author: Clontech (TaKaRa)

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SgrAI (6258) NdeI (5886) PfoI (5748) ScaI (5195) AflIII - PciI (3822) BstEII (1088) CsiI - SexAI * (1216) BsrGI (1283) 5' pMSCV primer (1333 .. 1355) EcoRI (1405) HpaI (1413) PaeR7I - XhoI (1417) BglII (1423) AgeI (1548) 3' pMSCV primer (1660 .. 1682) BlpI (1711) StuI (1732) BspEI (1857) BsmI (1869) BsaAI (2392) NcoI (2520) RsrII (2604) BamHI (2757) SalI (2763) AccI (2764) HindIII (2777) BspDI - ClaI (2784) pMSCVneo 6436 bp
SgrAI  (6258)
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  (5886)
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.
PfoI  (5748)
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.
ScaI  (5195)
1 site
A G T A C T T C A T G A
AflIII  (3822)
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  (3822)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
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.
CsiI  (1216)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
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.
EcoRI  (1405)
1 site
G A A T T C C T T A A G
HpaI  (1413)
1 site
G T T A A C C A A T T G
PaeR7I  (1417)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1417)
1 site
C T C G A G G A G C T C
BglII  (1423)
1 site
A G A T C T T C T A G A
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.
StuI  (1732)
1 site
A G G C C T T C C G G A
BspEI  (1857)
1 site
T C C G G A A G G C C T
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.
BsaAI  (2392)
1 site
Y A C G T R R T G C A Y
NcoI  (2520)
1 site
C C A T G G G G T A C C
RsrII  (2604)
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.
BamHI  (2757)
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.
SalI  (2763)
1 site
G T C G A C C A G C T G
AccI  (2764)
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  (2777)
1 site
A A G C T T T T C G A A
BspDI  (2784)
1 site
A T C G A T T A G C T A
ClaI  (2784)
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
4642 .. 5502  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4642 .. 5433  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4642 .. 5502  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5434 .. 5502  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4642 .. 5502  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
1951 .. 2754  =  804 bp
267 amino acids  =  29.3 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
1951 .. 2754  =  804 bp
267 amino acids  =  29.3 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
ori
3883 .. 4471  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3883 .. 4471  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
5' LTR
6435 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
5' LTR
6435 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
3' LTR
2829 .. 3343  =  515 bp
3' long terminal repeat from murine embryonic stem cell virus
3' LTR
2829 .. 3343  =  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
5503 .. 5607  =  105 bp
AmpR promoter
5503 .. 5607  =  105 bp
MCS
1405 .. 1428  =  24 bp
multiple cloning site
MCS
1405 .. 1428  =  24 bp
multiple cloning site
ORF:  1951 .. 2754  =  804 bp
ORF:  267 amino acids  =  29.3 kDa
ORF:  1718 .. 2131  =  414 bp
ORF:  137 amino acids  =  15.1 kDa
ORF:  2132 .. 2518  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  4772 .. 5038  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5888 .. 6184  =  297 bp
ORF:  98 amino acids  =  11.7 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:  2269 .. 2523  =  255 bp
ORF:  84 amino acids  =  9.6 kDa
ORF:  4642 .. 5502  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  5952 .. 6182  =  231 bp
ORF:  76 amino acids  =  7.5 kDa
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