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pTOL-hCMV-TET3G-Hygro Transactivator

Lentiviral vector with a hygromycin resistance marker for expression of the Tet-On® 3G doxycycline-dependent transactivator.

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pTOL-hCMV-TET3G-Hygro Transactivator Sequence and MappTOL-hCMV-TET3G-Hygro Transactivator.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SspI (11,153) FspI (10,571) AhdI (10,349) BstZ17I (9080) SexAI * (7496) PmeI (6681) Bsu36I (6288) SgrDI (11,270) KasI (1003) NarI (1004) SfoI (1005) PluTI (1007) BssHII (1077) FseI (1516) EcoNI (1536) BbvCI - Bpu10I (1790) AleI (1943) KflI - PpuMI (2300) cPPT/CTS BspDI - ClaI (2546) SnaBI (2846) HpaI (3157) BamHI (3176) PaeR7I - XhoI (3939) AarI (4292) BmgBI (4496) MluI (5554) pTOL-hCMV-TET3G-Hygro Transactivator 11,272 bp
SspI  (11,153)
1 site
A A T A T T T T A T A A
FspI  (10,571)
1 site
T G C G C A A C G C G T
AhdI  (10,349)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BstZ17I  (9080)
1 site
G T A T A C C A T A T G
SexAI  (7496)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
PmeI  (6681)
1 site
G T T T A A A C C A A A T T T G
Bsu36I  (6288)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
SgrDI  (11,270)
1 site
C G T C G A C G G C A G C T G C
KasI  (1003)
1 site
G G C G C C C C G C G G
NarI  (1004)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (1005)
1 site
G G C G C C C C G C G G
PluTI  (1007)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
BssHII  (1077)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
FseI  (1516)
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.
EcoNI  (1536)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BbvCI  (1790)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1790)
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.
AleI  (1943)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (2300)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (2300)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
BspDI  (2546)
1 site
A T C G A T T A G C T A
ClaI  (2546)
1 site
A T C G A T T A G C T A
SnaBI  (2846)
1 site
T A C G T A A T G C A T
HpaI  (3157)
1 site
G T T A A C C A A T T G
BamHI  (3176)
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.
PaeR7I  (3939)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (3939)
1 site
C T C G A G G A G C T C
AarI  (4292)
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.
BmgBI  (4496)
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.
MluI  (5554)
1 site
A C G C G T T G C G C A
HygR
4528 .. 5553  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
4528 .. 5553  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
7793 .. 8818  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
7793 .. 8818  =  1026 bp
341 amino acids  =  38.0 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
AmpR
10,276 .. 11,136  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,276 .. 11,067  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,276 .. 11,136  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   11,068 .. 11,136  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,276 .. 11,136  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
Tet-On® 3G
3188 .. 3934  =  747 bp
248 amino acids  =  27.7 kDa
Product: modified rtTA protein that binds tightly to
promoters containing the tet operator in the
presence of doxycycline
Tet-On® 3G
3188 .. 3934  =  747 bp
248 amino acids  =  27.7 kDa
Product: modified rtTA protein that binds tightly to
promoters containing the tet operator in the
presence of doxycycline
LTR
367 .. 1000  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
367 .. 1000  =  634 bp
long terminal repeat (LTR) from HIV-1
WPRE
5567 .. 6155  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
5567 .. 6155  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
9520 .. 10,105  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9520 .. 10,105  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3976 .. 4525  =  550 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3976 .. 4525  =  550 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
f1 ori
6973 .. 7401  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6973 .. 7401  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
7415 .. 7744  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
7415 .. 7744  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
2567 .. 2870  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2567 .. 2870  =  304 bp
human cytomegalovirus immediate early enhancer
RRE
1669 .. 1902  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1669 .. 1902  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
LTR (ΔU3)
6363 .. 6596  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
6363 .. 6596  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
bGH poly(A) signal
6703 .. 6927  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
6703 .. 6927  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
2871 .. 3074  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2871 .. 3074  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
HIV-1 Ψ
1047 .. 1172  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
1047 .. 1172  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
8948 .. 9069  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8948 .. 9069  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2394 .. 2509  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2394 .. 2509  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
11,137 .. 11,241  =  105 bp
AmpR promoter
11,137 .. 11,241  =  105 bp
SV40 ori
7595 .. 7730  =  136 bp
SV40 origin of replication
SV40 ori
7595 .. 7730  =  136 bp
SV40 origin of replication
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