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pRetroX-Tet-Off Advanced

Retroviral vector to be used in conjunction with a Tet-responsive vector for doxycycline-repressible expression of a gene.

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pRetroX-Tet-Off Advanced Sequence and MappRetroX-Tet-Off Advanced.dna
Map and Sequence File   
Sequence Author:  Clontech
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 SspI (7807) ScaI (7483) PvuI (7373) AlwNI (6526) SfiI (5816) BmtI (4892) NheI (4888) EcoRV (4746) BsgI (4655) PaeR7I - PspXI - XhoI (4605) RsrII (4291) CMV enhancer AscI (542) PshAI (676) BstEII (1252) Bpu10I (1455) BglII (1576) SbfI (2230) NotI (2243) AgeI (2250) BsiWI (2262) PacI (2273) BamHI (2278) XcmI (2292) NruI (2567) MfeI (2804) FseI (2911) EcoRI (3037) AarI (3399) PflMI (3513) BmgBI (3603) BstXI (3646) pRetroX-Tet-Off Advanced 8152 bp
SspI  (7807)
1 site
A A T A T T T T A T A A
ScaI  (7483)
1 site
A G T A C T T C A T G A
PvuI  (7373)
1 site
C G A T C G G C T A G C
AlwNI  (6526)
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.
SfiI  (5816)
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.
BmtI  (4892)
1 site
G C T A G C C G A T C G
NheI  (4888)
1 site
G C T A G C C G A T C G
EcoRV  (4746)
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.
BsgI  (4655)
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).
PaeR7I  (4605)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4605)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4605)
1 site
C T C G A G G A G C T C
RsrII  (4291)
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.
AscI  (542)
1 site
G G C G C G C C C C G C G C G G
PshAI  (676)
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.
BstEII  (1252)
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.
Bpu10I  (1455)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
BglII  (1576)
1 site
A G A T C T T C T A G A
SbfI  (2230)
1 site
C C T G C A G G G G A C G T C C
NotI  (2243)
1 site
G C G G C C G C C G C C G G C G
AgeI  (2250)
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.
BsiWI  (2262)
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.
PacI  (2273)
1 site
T T A A T T A A A A T T A A T T
BamHI  (2278)
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.
XcmI  (2292)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
NruI  (2567)
1 site
T C G C G A A G C G C T
MfeI  (2804)
1 site
C A A T T G G T T A A C
FseI  (2911)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
EcoRI  (3037)
1 site
G A A T T C C T T A A G
AarI  (3399)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
PflMI  (3513)
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.
BmgBI  (3603)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BstXI  (3646)
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.
AmpR
6930 .. 7790  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   6930 .. 7721  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6930 .. 7790  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7722 .. 7790  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6930 .. 7790  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
3647 .. 4441  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3647 .. 4441  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 1:  TetR  
   2287 .. 2904  =  618 bp
   206 amino acids  =  23.3 kDa
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 2:  
   2905 .. 2910  =  6 bp
   2 amino acids  =  172.2 Da
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 3:  VP16 minimal transcriptional activation domain  
   2911 .. 2946  =  36 bp
   12 amino acids  =  1.4 kDa
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 4:  
   2947 .. 2949  =  3 bp
   1 amino acid  =  115.1 Da
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 5:  VP16 minimal transcriptional activation domain  
   2950 .. 2985  =  36 bp
   12 amino acids  =  1.4 kDa
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 6:  
   2986 .. 2988  =  3 bp
   1 amino acid  =  115.1 Da
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 7:  VP16 minimal transcriptional activation domain  
   2989 .. 3024  =  36 bp
   12 amino acids  =  1.3 kDa
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
   Segment 8:  
   3025 .. 3033  =  9 bp
   2 amino acids  =  172.2 Da
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
tTA-Advanced
2287 .. 3033  =  747 bp
248 amino acids  =  27.7 kDa
8 segments
Product: improved tetracycline-controlled
transactivator
binds to a Tet-responsive element in the absence
but not the presence of doxycycline
ori
6171 .. 6759  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6171 .. 6759  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3060 .. 3633  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3060 .. 3633  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
3' LTR (ΔU3)
4858 .. 5283  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
3' LTR (ΔU3)
4858 .. 5283  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
gag (truncated)
1151 .. 1567  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
1151 .. 1567  =  417 bp
truncated MMLV gag gene lacking the start codon
CMV enhancer
8081 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
8081 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5549 .. 5878  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5549 .. 5878  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
1604 .. 1907  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
1604 .. 1907  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
309 .. 512  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
309 .. 512  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
1908 .. 2111  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
1908 .. 2111  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
MMLV Ψ
751 .. 950  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
MMLV Ψ
751 .. 950  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
5' LTR (truncated)
513 .. 688  =  176 bp
truncated long terminal repeat from Moloney murine
sarcoma virus
5' LTR (truncated)
513 .. 688  =  176 bp
truncated long terminal repeat from Moloney murine
sarcoma virus
AmpR promoter
7791 .. 7895  =  105 bp
AmpR promoter
7791 .. 7895  =  105 bp
SV40 ori
5729 .. 5864  =  136 bp
SV40 origin of replication
SV40 ori
5729 .. 5864  =  136 bp
SV40 origin of replication
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