pRetroX-Tet3G

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

Sequence Author: Clontech (TaKaRa)

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SspI (7791) ScaI (7467) PvuI (7357) AlwNI (6510) SfiI (5800) BmtI (4876) NheI (4872) EcoRV (4730) BsgI (4639) PaeR7I - PspXI - XhoI (4589) RsrII (4275) NaeI (4261) 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 (2295) NruI (2570) MfeI (2807) EcoRI (3037) PaqCI (3384) PflMI (3498) BmgBI (3587) BstXI (3630) NgoMIV (4259) pRetroX-Tet3G 8136 bp
SspI  (7791)
1 site
A A T A T T T T A T A A
ScaI  (7467)
1 site
A G T A C T T C A T G A
PvuI  (7357)
1 site
C G A T C G G C T A G C
AlwNI  (6510)
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  (5800)
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  (4876)
1 site
G C T A G C C G A T C G
NheI  (4872)
1 site
G C T A G C C G A T C G
EcoRV  (4730)
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  (4639)
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  (4589)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4589)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4589)
1 site
C T C G A G G A G C T C
RsrII  (4275)
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.
NaeI  (4261)
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.
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

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.
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
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  (2295)
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  (2570)
1 site
T C G C G A A G C G C T
MfeI  (2807)
1 site
C A A T T G G T T A A C
EcoRI  (3037)
1 site
G A A T T C C T T A A G
PaqCI  (3384)
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 PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PflMI  (3498)
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  (3587)
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  (3630)
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.
NgoMIV  (4259)
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.
AmpR
6914 .. 7774  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   6914 .. 7705  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6914 .. 7774  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   7706 .. 7774  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6914 .. 7774  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
3631 .. 4425  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
3631 .. 4425  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
Tet-On® 3G
2290 .. 3036  =  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
2290 .. 3036  =  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
ori
6155 .. 6743  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6155 .. 6743  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
3045 .. 3617  =  573 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
3045 .. 3617  =  573 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
3' LTR (ΔU3)
4842 .. 5267  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
3' LTR (ΔU3)
4842 .. 5267  =  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
8065 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
8065 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5533 .. 5862  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5533 .. 5862  =  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
7775 .. 7879  =  105 bp
AmpR promoter
7775 .. 7879  =  105 bp
SV40 ori
5713 .. 5848  =  136 bp
SV40 origin of replication
SV40 ori
5713 .. 5848  =  136 bp
SV40 origin of replication
ORF:  2290 .. 3036  =  747 bp
ORF:  248 amino acids  =  27.7 kDa
ORF:  3631 .. 4425  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  2447 .. 2749  =  303 bp
ORF:  100 amino acids  =  11.1 kDa
ORF:  3803 .. 4189  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  7044 .. 7310  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  838 .. 1098  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  3940 .. 4194  =  255 bp
ORF:  84 amino acids  =  9.6 kDa
ORF:  2742 .. 3023  =  282 bp
ORF:  93 amino acids  =  9.6 kDa
ORF:  5091 .. 5381  =  291 bp
ORF:  96 amino acids  =  10.5 kDa
ORF:  6914 .. 7774  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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