pSIREN-RetroQ-TetH

Linearized retroviral vector with a hygromycin resistance marker for inserting an shRNA cassette that will be expressed from a Tet-responsive promoter.

Sequence Author: Clontech (TaKaRa)

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<BamHI> (7388) BglII (7033) U6 forward sequencing primer (6956 .. 6983) BstEII (6709) AfeI (6545) ApoI (6494) AscI (5999) SnaBI (5741) CMV enhancerSspI (5111) XmnI (4906) FspI (4529) <EcoRI> (0) AgeI (120) BsmI (441) AsiSI (891) RsrII (935) SacII (1307) BbvCI (1592) AleI (1778) BspDI - ClaI (1865) EcoRV (2050) PvuII (2093) SfiI (3120) AvrII (3167) BspQI - SapI (3298) AflIII - PciI (3414) pSIREN-RetroQ-TetH 7388 bp
End  (7388)
0 sites
BglII  (7033)
1 site
A G A T C T T C T A G A
BstEII  (6709)
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.
AfeI  (6545)
1 site
A G C G C T T C G C G A
ApoI  (6494)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
AscI  (5999)
1 site
G G C G C G C C C C G C G C G G
SnaBI  (5741)
1 site
T A C G T A A T G C A T
SspI  (5111)
1 site
A A T A T T T T A T A A
XmnI  (4906)
1 site
G A A N N N N T T C C T T N N N N A A G
FspI  (4529)
1 site
T G C G C A A C G C G T
Start  (0)
0 sites
AgeI  (120)
1 site
A C C G G T T G G C C A
BsmI  (441)
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.
AsiSI  (891)
1 site
G C G A T C G C C G C T A G C G
RsrII  (935)
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  (1307)
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.
BbvCI  (1592)
1 site
C C T C A G C G G A G T C G
AleI  (1778)
1 site
C A C N N N N G T G G T G N N N N C A C
BspDI  (1865)
1 site
A T C G A T T A G C T A
ClaI  (1865)
1 site
A T C G A T T A G C T A
EcoRV  (2050)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
PvuII  (2093)
1 site
C A G C T G G T C G A C
SfiI  (3120)
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  (3167)
1 site
C C T A G G G G A T C C
BspQI  (3298)
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  (3298)
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  (3414)
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  (3414)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
U6 forward sequencing primer
28-mer  /  64% GC
1 binding site
6956 .. 6983  =  28 annealed bases
Tm  =  68°C
HygR
527 .. 1552  =  1026 bp
341 amino acids  =  38.0 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
527 .. 1552  =  1026 bp
341 amino acids  =  38.0 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
4234 .. 5094  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4234 .. 5025  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4234 .. 5094  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5026 .. 5094  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4234 .. 5094  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3475 .. 4063  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3475 .. 4063  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
PGK promoter
10 .. 509  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
10 .. 509  =  500 bp
mouse phosphoglycerate kinase 1 promoter
3' LTR (ΔU3)
2162 .. 2587  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
3' LTR (ΔU3)
2162 .. 2587&#