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pMSCV16URP-U6-sh-HTS6-UbiC-TagRFP-2A-Puro

Lentiviral vector derived from murine embryonic stem cell virus, for barcoded shRNA libraries, with TagRFP and puromycin resistance markers.

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 SgrAI (7424) SspI (6685) XmnI (6480) ScaI (6361) AflIII - PciI (4988) HindIII (4757) AvrII (4741) SfiI (4694) HincII (3669) BbvCI (209) AscI (369) SpeI (661) BglII (1410) BbsI - BpiI (1683) cloning site BbsI - BpiI (1709) PaeR7I - PspXI - XhoI (1732) BsgI (1777) EcoRI (1849) AgeI (1858) BstBI (1867) AleI (2284) PflMI (2889) PshAI (2923) BamHI (2995) BsiWI (3117) BspEI * (3174) RsrII (3177) SalI (3667) pMSCV16URP-U6-sh-HTS6-UbiC-TagRFP-2A-Puro 7602 bp
SgrAI  (7424)
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.
SspI  (6685)
1 site
A A T A T T T T A T A A
XmnI  (6480)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (6361)
1 site
A G T A C T T C A T G A
AflIII  (4988)
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  (4988)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
HindIII  (4757)
1 site
A A G C T T T T C G A A
AvrII  (4741)
1 site
C C T A G G G G A T C C
SfiI  (4694)
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.
HincII  (3669)
1 site
G T Y R A C C A R Y T G
BbvCI  (209)
1 site
C C T C A G C G G A G T C G
AscI  (369)
1 site
G G C G C G C C C C G C G C G G
SpeI  (661)
1 site
A C T A G T T G A T C A
BglII  (1410)
1 site
A G A T C T T C T A G A
BbsI  (1683)
2 sites
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.
BpiI  (1683)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
BbsI  (1709)
2 sites
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.
BpiI  (1709)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
PaeR7I  (1732)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1732)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1732)
1 site
C T C G A G G A G C T C
BsgI  (1777)
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).
EcoRI  (1849)
1 site
G A A T T C C T T A A G
AgeI  (1858)
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.
BstBI  (1867)
1 site
T T C G A A A A G C T T
AleI  (2284)
1 site
C A C N N N N G T G G T G N N N N C A C
PflMI  (2889)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
PshAI  (2923)
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.
BamHI  (2995)
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.
BsiWI  (3117)
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  (3174)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
RsrII  (3177)
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.
SalI  (3667)
1 site
G T C G A C C A G C T G
AmpR
5808 .. 6668  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5808 .. 6599  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5808 .. 6668  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6600 .. 6668  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5808 .. 6668  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagRFP
2284 .. 2994  =  711 bp
237 amino acids  =  26.7 kDa
Product: monomeric derivative of red fluorescent
protein from Entacmaea quadricolor (Merzlyak et
al., 2007)
mammalian codon-optimized
TagRFP
2284 .. 2994  =  711 bp
237 amino acids  =  26.7 kDa
Product: monomeric derivative of red fluorescent
protein from Entacmaea quadricolor (Merzlyak et
al., 2007)
mammalian codon-optimized
PuroR
3061 .. 3660  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
3061 .. 3660  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
5049 .. 5637  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5049 .. 5637  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
5' LTR
7601 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
5' LTR
7601 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
3' LTR (ΔU3)
3736 .. 4161  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
3' LTR (ΔU3)
3736 .. 4161  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
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
UbiC promoter
1872 .. 2271  =  400 bp
human ubiquitin C promoter
UbiC promoter
1872 .. 2271  =  400 bp
human ubiquitin C promoter
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
SV40 promoter
4427 .. 4756  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
4427 .. 4756  =  330 bp
SV40 enhancer and early promoter
U6 promoter
1438 .. 1678  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
1438 .. 1678  =  241 bp
RNA polymerase III promoter for human U6 snRNA
AmpR promoter
6669 .. 6773  =  105 bp
AmpR promoter
6669 .. 6773  =  105 bp
T2A
3001 .. 3054  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
3001 .. 3054  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
cloning site
1684 .. 1713  =  30 bp
shRNA cloning site
cloning site
1684 .. 1713  =  30 bp
shRNA cloning site
SV40 ori
4607 .. 4742  =  136 bp
SV40 origin of replication
SV40 ori
4607 .. 4742  =  136 bp
SV40 origin of replication
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