pSIREN-RetroQ-DsRed-Express

Linearized retroviral vector for inserting an shRNA cassette that will be co-expressed with DsRed-Express.

Sequence Author: Clontech (TaKaRa)

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<BamHI> (6543) U6 forward sequencing primer (6468 .. 6491) BglII (6285) Bpu10I (6164) ApoI (5746) EagI (5598) PshAI (5385) AscI (5251) CMV enhancerSspI (4364) XmnI (4159) ScaI (4040) PvuI (3930) <EcoRI> (0) AgeI (605) FspAI (666) SbfI (958) PflMI* (1136) BstXI (1160) BsgI (1197) AleI (1245) PaeR7I - XhoI (1296) EcoRV (1303) SalI (1538) AccI (1539) SfiI (2373) AvrII (2420) HindIII (2436) BspQI - SapI (2551) AflIII - PciI (2667) AlwNI (3083) pSIREN-RetroQ-DsRed-Express 6543 bp
End  (6543)
0 sites
BglII  (6285)
1 site
A G A T C T T C T A G A
Bpu10I  (6164)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
ApoI  (5746)
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.
EagI  (5598)
1 site
C G G C C G G C C G G C
PshAI  (5385)
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.
AscI  (5251)
1 site
G G C G C G C C C C G C G C G G
SspI  (4364)
1 site
A A T A T T T T A T A A
XmnI  (4159)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (4040)
1 site
A G T A C T T C A T G A
PvuI  (3930)
1 site
C G A T C G G C T A G C
Start  (0)
0 sites
AgeI  (605)
1 site
A C C G G T T G G C C A
FspAI  (666)
1 site
R T G C G C A Y Y A C G C G T R
SbfI  (958)
1 site
C C T G C A G G G G A C G T C C
PflMI  (1136)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BstXI  (1160)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BsgI  (1197)
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).
AleI  (1245)
1 site
C A C N N N N G T G G T G N N N N C A C
PaeR7I  (1296)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1296)
1 site
C T C G A G G A G C T C
EcoRV  (1303)
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.
SalI  (1538)
1 site
G T C G A C C A G C T G
AccI  (1539)
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.
SfiI  (2373)
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  (2420)
1 site
C C T A G G G G A T C C
HindIII  (2436)
1 site
A A G C T T T T C G A A
BspQI  (2551)
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  (2551)
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  (2667)
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  (2667)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AlwNI  (3083)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
U6 forward sequencing primer
24-mer  /  38% GC
1 binding site
6468 .. 6491  =  24 annealed bases
Tm  =  55°C
AmpR
3487 .. 4347  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3487 .. 4278  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3487 .. 4347  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4279 .. 4347  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3487 .. 4347  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
DsRed-Express
618 .. 1295  =  678 bp
225 amino acids  =  25.7 kDa
Product: rapidly maturing tetrameric variant of DsRed fluorescent protein
mammalian codon-optimized
DsRed-Express
618 .. 1295  =  678 bp
225 amino acids  =  25.7 kDa
Product: rapidly maturing tetrameric variant of DsRed fluorescent protein
mammalian codon-optimized
ori
2728 .. 3316  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2728 .. 3316  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
3' LTR (ΔU3)
1415 .. 1840  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
3' LTR (ΔU3)
1415 .. 1840  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
gag (truncated)
5860 .. 6276  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
5860 .. 6276  =  417 bp
truncated MMLV gag gene lacking the start codon
CMV enhancer
4638 .. 5017  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4638 .. 5017  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
2106 .. 2435  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2106 .. 2435  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
66 .. 369  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
66 .. 369  =  304 bp
human cytomegalovirus immediate early enhancer
U6 promoter
6300 .. 6540  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6300 .. 6540  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
370 .. 573  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
370 .. 573  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
5018 .. 5221  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
5018 .. 5221  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
MMLV Ψ
5460 .. 5659  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
MMLV Ψ
5460 .. 5659  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
5' LTR (truncated)
5222 .. 5397  =  176 bp
truncated long terminal repeat from Moloney murine sarcoma virus
5' LTR (truncated)
5222 .. 5397  =  176 bp
truncated long terminal repeat from Moloney murine sarcoma virus
AmpR promoter
4348 .. 4452  =  105 bp
AmpR promoter
4348 .. 4452  =  105 bp
SV40 ori
2286 .. 2421  =  136 bp
SV40 origin of replication
SV40 ori
2286 .. 2421  =  136 bp
SV40 origin of replication
ORF:  3617 .. 3883  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  618 .. 1295  =  678 bp
ORF:  225 amino acids  =  25.7 kDa
ORF:  1664 .. 1954  =  291 bp
ORF:  96 amino acids  =  10.5 kDa
ORF:  3487 .. 4347  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  5547 .. 5807  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
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