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Plasmid Files

pSIREN-RetroQ-TetP

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

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pSIREN-RetroQ-TetP Sequence and MappSIREN-RetroQ-TetP.dna
Map and Sequence File   
Sequence Author:  Clontech
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 < BamHI > (6536) BglII (6181) U6 forward sequencing primer (6104 .. 6131) AfeI (5693) ApoI (5642) PshAI (5281) AscI (5147) SnaBI (4889) BtgZI (4883) CMV enhancer SspI (4259) XmnI (4054) ScaI (3935) PvuI (3825) FspI (3677) < EcoRI > (0) AgeI (120) BlpI (283) BsmI (441) BfuAI - BspMI (519) BsiWI (586) RsrII (646) SacII (744) DraIII (1101) PaeR7I - XhoI (1191) EcoRV (1198) PvuII (1241) NheI (1340) BmtI (1344) SalI (1433) AccI (1434) SfiI (2268) AvrII (2315) BspQI - SapI (2446) AflIII - PciI (2562) pSIREN-RetroQ-TetP 6536 bp
End  (6536)
0 sites
BglII  (6181)
1 site
A G A T C T T C T A G A
AfeI  (5693)
1 site
A G C G C T T C G C G A
ApoI  (5642)
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.
PshAI  (5281)
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  (5147)
1 site
G G C G C G C C C C G C G C G G
SnaBI  (4889)
1 site
T A C G T A A T G C A T
BtgZI  (4883)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
SspI  (4259)
1 site
A A T A T T T T A T A A
XmnI  (4054)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3935)
1 site
A G T A C T T C A T G A
PvuI  (3825)
1 site
C G A T C G G C T A G C
FspI  (3677)
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

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BlpI  (283)
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  (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.
BfuAI  (519)
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  (519)
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.
BsiWI  (586)
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  (646)
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  (744)
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  (1101)
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.
PaeR7I  (1191)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1191)
1 site
C T C G A G G A G C T C
EcoRV  (1198)
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  (1241)
1 site
C A G C T G G T C G A C
NheI  (1340)
1 site
G C T A G C C G A T C G
BmtI  (1344)
1 site
G C T A G C C G A T C G
SalI  (1433)
1 site
G T C G A C C A G C T G
AccI  (1434)
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  (2268)
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  (2315)
1 site
C C T A G G G G A T C C
BspQI  (2446)
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  (2446)
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  (2562)
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  (2562)
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
6104 .. 6131  =  28 annealed bases
Tm  =  68°C
AmpR
3382 .. 4242  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3382 .. 4173  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3382 .. 4242  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4174 .. 4242  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3382 .. 4242  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
530 .. 1129  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
530 .. 1129  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
2623 .. 3211  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2623 .. 3211  =  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)
1310 .. 1735  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
3' LTR (ΔU3)
1310 .. 1735  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
gag (truncated)
5756 .. 6172  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
5756 .. 6172  =  417 bp
truncated MMLV gag gene lacking the start codon
CMV enhancer
4533 .. 4913  =  381 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4533 .. 4913  =  381 bp
human cytomegalovirus immediate early enhancer
TREmod/U6 promoter
6202 .. 6533  =  332 bp
modified Tet-responsive promoter, consisting of
seven repeats of the tet operator fused to a portion
of the human U6 promoter
TREmod/U6 promoter
6202 .. 6533  =  332 bp
modified Tet-responsive promoter, consisting of
seven repeats of the tet operator fused to a portion
of the human U6 promoter
SV40 promoter
2001 .. 2330  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2001 .. 2330  =  330 bp
SV40 enhancer and early promoter
CMV promoter
4914 .. 5117  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
4914 .. 5117  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
MMLV Ψ
5356 .. 5555  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
MMLV Ψ
5356 .. 5555  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
5' LTR (truncated)
5118 .. 5293  =  176 bp
truncated long terminal repeat from Moloney murine
sarcoma virus
5' LTR (truncated)
5118 .. 5293  =  176 bp
truncated long terminal repeat from Moloney murine
sarcoma virus
AmpR promoter
4243 .. 4347  =  105 bp
AmpR promoter
4243 .. 4347  =  105 bp
SV40 ori
2181 .. 2316  =  136 bp
SV40 origin of replication
SV40 ori
2181 .. 2316  =  136 bp
SV40 origin of replication
tet operator
6210 .. 6228  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6210 .. 6228  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6246 .. 6264  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6246 .. 6264  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6281 .. 6299  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6281 .. 6299  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6317 .. 6335  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6317 .. 6335  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6353 .. 6371  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6353 .. 6371  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6388 .. 6406  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6388 .. 6406  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6424 .. 6442  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
6424 .. 6442  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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