pRetroX-TRE3G

Retroviral vector for doxycycline-inducible expression of a gene in cells containing the Tet-On® 3G transactivator protein.

Sequence Author: Clontech (TaKaRa)

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CMV enhancer SspI (6255) XmnI (6050) ScaI (5931) PvuI (5821) FspI (5673) PciI (4558) BspQI - SapI (4442) AvrII (4311) SfiI (4264) AccI (3430) SalI (3429) BmtI (3340) NheI (3336) BtgZI (279) SnaBI (285) AscI (543) PshAI (677) AfeI (1089) BglII (1577) PspXI (1605) BamHI (1958) BspDI - ClaI (1965) MluI (1970) NotI (1977) MreI - NgoMIV - SgrAI (1984) NaeI (1986) PspOMI (1990) ApaI (1994) EcoRI (1996) AgeI (2116) BlpI (2279) BsmI (2437) BfuAI - BspMI (2515) BsiWI (2582) RsrII (2642) SacII (2740) DraIII (3097) EcoRV (3194) PvuII (3237) pRetroX-TRE3G 6600 bp
SspI  (6255)
1 site
A A T A T T T T A T A A
XmnI  (6050)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (5931)
1 site
A G T A C T T C A T G A
PvuI  (5821)
1 site
C G A T C G G C T A G C
FspI  (5673)
1 site
T G C G C A A C G C G T
PciI  (4558)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4442)
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  (4442)
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.
AvrII  (4311)
1 site
C C T A G G G G A T C C
SfiI  (4264)
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.
AccI  (3430)
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.
SalI  (3429)
1 site
G T C G A C C A G C T G
BmtI  (3340)
1 site
G C T A G C C G A T C G
NheI  (3336)
1 site
G C T A G C C G A T C G
BtgZI  (279)
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.
SnaBI  (285)
1 site
T A C G T A A T G C A T
AscI  (543)
1 site
G G C G C G C C C C G C G C G G
PshAI  (677)
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.
AfeI  (1089)
1 site
A G C G C T T C G C G A
BglII  (1577)
1 site
A G A T C T T C T A G A
PspXI  (1605)
1 site
V C T C G A G B B G A G C T C V
BamHI  (1958)
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.
BspDI  (1965)
1 site
A T C G A T T A G C T A
ClaI  (1965)
1 site
A T C G A T T A G C T A
MluI  (1970)
1 site
A C G C G T T G C G C A
NotI  (1977)
1 site
G C G G C C G C C G C C G G C G
MreI  (1984)
1 site
C G C C G G C G G C G G C C G C
NgoMIV  (1984)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
SgrAI  (1984)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
NaeI  (1986)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
PspOMI  (1990)
1 site
G G G C C C C C C G G G
ApaI  (1994)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRI  (1996)
1 site
G A A T T C C T T A A G
AgeI  (2116)
1 site
A C C G G T T G G C C A
BlpI  (2279)
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  (2437)
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  (2515)
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  (2515)
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  (2582)
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  (2642)
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  (2740)
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  (3097)
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.
EcoRV  (3194)
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  (3237)
1 site
C A G C T G G T C G A C
AmpR
5378 .. 6238  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   5378 .. 6169  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5378 .. 6238  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   6170 .. 6238  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5378 .. 6238  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
PuroR
2526 .. 3125  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
2526 .. 3125  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
4619 .. 5207  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4619 .. 5207  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
PGK promoter
2006 .. 2505  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
2006 .. 2505  =  500 bp
mouse phosphoglycerate kinase 1 promoter
3' LTR (ΔU3)
3306 .. 3731  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
3' LTR (ΔU3)
3306 .. 3731  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
gag (truncated)
1152 .. 1568  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
1152 .. 1568  =  417 bp
truncated MMLV gag gene lacking the start codon
CMV enhancer
6529 .. 309  =  381 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
6529 .. 309  =  381 bp
human cytomegalovirus immediate early enhancer
TRE3GV promoter
1611 .. 1957  =  347 bp
3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On 3G; optimized for retroviral and lentiviral vectors
TRE3GV promoter
1611 .. 1957  =  347 bp
3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On 3G; optimized for retroviral and lentiviral vectors
SV40 promoter
3997 .. 4326  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
3997 .. 4326  =  330 bp
SV40 enhancer and early promoter
CMV promoter
310 .. 513  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
310 .. 513  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
MMLV Ψ
752 .. 951  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
MMLV Ψ
752 .. 951  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
5' LTR (truncated)
514 .. 689  =  176 bp
truncated long terminal repeat from Moloney murine sarcoma virus
5' LTR (truncated)
514 .. 689  =  176 bp
truncated long terminal repeat from Moloney murine sarcoma virus
AmpR promoter
6239 .. 6343  =  105 bp
AmpR promoter
6239 .. 6343  =  105 bp
MCS
1958 .. 2001  =  44 bp
multiple cloning site
MCS
1958 .. 2001  =  44 bp
multiple cloning site
SV40 ori
4177 .. 4312  =  136 bp
SV40 origin of replication
SV40 ori
4177 .. 4312  =  136 bp
SV40 origin of replication
tet operator
1616 .. 1634  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1616 .. 1634  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1652 .. 1670  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1652 .. 1670  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1688 .. 1706  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1688 .. 1706  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1724 .. 1742  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1724 .. 1742  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1760 .. 1778  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1760 .. 1778  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1796 .. 1814  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1796 .. 1814  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1832 .. 1850  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1832 .. 1850  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ORF:  1585 .. 1866  =  282 bp
ORF:  93 amino acids  =  11.1 kDa
ORF:  2286 .. 3125  =  840 bp
ORF:  279 amino acids  =  30.1 kDa
ORF:  5508 .. 5774  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2451 .. 3176  =  726 bp
ORF:  241 amino acids  =  24.8 kDa
ORF:  3555 .. 3845  =  291 bp
ORF:  96 amino acids  =  10.5 kDa
ORF:  839 .. 1099  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  1838 .. 2113  =  276 bp
ORF:  91 amino acids  =  10.5 kDa
ORF:  5378 .. 6238  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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