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Plasmid Files

pQC-tTS-IN

Retroviral vector for expressing tTS, which represses expression from Tet-responsive promoters in the absence of doxycycline.

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pQC-tTS-IN Sequence and MappQC-tTS-IN.dna
Map and Sequence File   
Sequence Author:  Clontech
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 ScaI (7557) PvuI (7447) AlwNI (6600) StuI (5936) SfiI (5890) EcoRV (4820) PaeR7I - PspXI - XhoI (4679) RsrII (4365) NaeI (4351) NgoMIV (4349) CMV enhancer AscI (542) PshAI (676) BstEII (1252) Bpu10I (1455) BglII (1576) SbfI (2230) NotI (2243) AgeI - SgrAI (2250) BamHI (3098) EcoRI (3111) PspOMI (3247) ApaI (3251) AarI (3473) PflMI (3587) BmgBI (3677) pQC-tTS-IN 8226 bp
ScaI  (7557)
1 site
A G T A C T T C A T G A
PvuI  (7447)
1 site
C G A T C G G C T A G C
AlwNI  (6600)
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.
StuI  (5936)
1 site
A G G C C T T C C G G A
SfiI  (5890)
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.
EcoRV  (4820)
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.
PaeR7I  (4679)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4679)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4679)
1 site
C T C G A G G A G C T C
RsrII  (4365)
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  (4351)
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.
NgoMIV  (4349)
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.
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.
SgrAI  (2250)
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.
BamHI  (3098)
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.
EcoRI  (3111)
1 site
G A A T T C C T T A A G
PspOMI  (3247)
1 site
G G G C C C C C C G G G
ApaI  (3251)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AarI  (3473)
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  (3587)
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  (3677)
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.
AmpR
7004 .. 7864  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   7004 .. 7795  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7004 .. 7864  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7796 .. 7864  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7004 .. 7864  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
tTS
2262 .. 3110  =  849 bp
282 amino acids  =  31.9 kDa
   Segment 1:  TetR  
   2262 .. 2894  =  633 bp
   211 amino acids  =  23.6 kDa
Product: fusion of a hybrid tetracycline repressor
TetR with a nuclear localization signal and a KRAB
silencing domain
silences Tet-responsive promoters in the absence
but not the presence of doxycycline
tTS
2262 .. 3110  =  849 bp
282 amino acids  =  31.9 kDa
   Segment 2:  NLS  
   2895 .. 2915  =  21 bp
   7 amino acids  =  897.2 Da
Product: fusion of a hybrid tetracycline repressor
TetR with a nuclear localization signal and a KRAB
silencing domain
silences Tet-responsive promoters in the absence
but not the presence of doxycycline
tTS
2262 .. 3110  =  849 bp
282 amino acids  =  31.9 kDa
   Segment 3:  KRAB silencing domain  
   2916 .. 3110  =  195 bp
   64 amino acids  =  7.4 kDa
Product: fusion of a hybrid tetracycline repressor
TetR with a nuclear localization signal and a KRAB
silencing domain
silences Tet-responsive promoters in the absence
but not the presence of doxycycline
tTS
2262 .. 3110  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
Product: fusion of a hybrid tetracycline repressor
TetR with a nuclear localization signal and a KRAB
silencing domain
silences Tet-responsive promoters in the absence
but not the presence of doxycycline
NeoR/KanR
3721 .. 4515  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3721 .. 4515  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ori
6245 .. 6833  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6245 .. 6833  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3134 .. 3707  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3134 .. 3707  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
3' LTR (ΔU3)
4932 .. 5357  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
3' LTR (ΔU3)
4932 .. 5357  =  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
8155 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
8155 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5623 .. 5952  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5623 .. 5952  =  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
7865 .. 7969  =  105 bp
AmpR promoter
7865 .. 7969  =  105 bp
SV40 ori
5803 .. 5938  =  136 bp
SV40 origin of replication
SV40 ori
5803 .. 5938  =  136 bp
SV40 origin of replication
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