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pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS

Vector encoding a single-chain anti-GCN4 peptide antibody fused to superfolder GFP, the VP64 activation domain, and the GB1 solubilization domain.

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pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS Sequence and MappHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS.dna
Map and Sequence File   
Sequence Author:  Vale Lab / Addgene #60904
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 NotI (9846) SpeI (9776) BstBI (9287) BstZ17I (9115) PspXI (9086) MscI (8836) BamHI (8644) BlpI (8270) AarI (7892) SmaI (7768) TspMI - XmaI (7766) PflFI - Tth111I (7759) BspDI * - ClaI * (7501) KflI (7230) BbvCI (6720) NruI * (6134) BssHII (6012) PmlI (5591) XcmI (5205) NheI (357) BmtI (361) NgoMIV (390) NaeI (392) AfeI (485) EcoNI (615) PfoI (779) PvuI (1449) AhdI (1818) SfiI (3163) PshAI (4040) PflMI (4434) HpaI (4998) pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS 10,074 bp
NotI  (9846)
1 site
G C G G C C G C C G C C G G C G
SpeI  (9776)
1 site
A C T A G T T G A T C A
BstBI  (9287)
1 site
T T C G A A A A G C T T
BstZ17I  (9115)
1 site
G T A T A C C A T A T G
PspXI  (9086)
1 site
V C T C G A G B B G A G C T C V
MscI  (8836)
1 site
T G G C C A A C C G G T
BamHI  (8644)
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.
BlpI  (8270)
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.
AarI  (7892)
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.
SmaI  (7768)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (7766)
1 site
C C C G G G G G G C C C
XmaI  (7766)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
PflFI  (7759)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (7759)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BspDI  (7501)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (7501)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
KflI  (7230)
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.
BbvCI  (6720)
1 site
C C T C A G C G G A G T C G
NruI  (6134)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BssHII  (6012)
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.
PmlI  (5591)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
XcmI  (5205)
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.
NheI  (357)
1 site
G C T A G C C G A T C G
BmtI  (361)
1 site
G C T A G C C G A T C G
NgoMIV  (390)
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.
NaeI  (392)
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.
AfeI  (485)
1 site
A G C G C T T C G C G A
EcoNI  (615)
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.
PfoI  (779)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PvuI  (1449)
1 site
C G A T C G G C T A G C
AhdI  (1818)
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.
SfiI  (3163)
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.
PshAI  (4040)
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.
PflMI  (4434)
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.
HpaI  (4998)
1 site
G T T A A C C A A T T G
AmpR
1031 .. 1891  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1031 .. 1099  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1031 .. 1891  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1100 .. 1891  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1031 .. 1891  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
scFv GCN4
7811 .. 8551  =  741 bp
247 amino acids  =  25.8 kDa
Product: single-chain Fv antibody fragment against
the GCN4 peptide
scFv GCN4
7811 .. 8551  =  741 bp
247 amino acids  =  25.8 kDa
Product: single-chain Fv antibody fragment against
the GCN4 peptide
superfolder GFP
8669 .. 9379  =  711 bp
237 amino acids  =  26.7 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
8669 .. 9379  =  711 bp
237 amino acids  =  26.7 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
LTR
5302 .. 5935  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
5302 .. 5935  =  634 bp
long terminal repeat (LTR) from HIV-1
ori
2062 .. 2650  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2062 .. 2650  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
SV40 promoter
2896 .. 3225  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2896 .. 3225  =  330 bp
SV40 enhancer and early promoter
RRE
6599 .. 6832  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
6599 .. 6832  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
LTR (ΔU3)
9935 .. 94  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
9935 .. 94  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
SV40 promoter
7526 .. 7736  =  211 bp
SV40 early promoter
SV40 promoter
7526 .. 7736  =  211 bp
SV40 early promoter
GB1
9593 .. 9754  =  162 bp
54 amino acids  =  6.0 kDa
Product: B1 domain of Streptococcal protein G  
effective as a solubilizing fusion partner (Cheng and
Patel, 2004)
GB1
9593 .. 9754  =  162 bp
54 amino acids  =  6.0 kDa
Product: B1 domain of Streptococcal protein G  
effective as a solubilizing fusion partner (Cheng and
Patel, 2004)
VP64
9413 .. 9562  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
VP64
9413 .. 9562  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
SV40 poly(A) signal
4999 .. 5133  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4999 .. 5133  =  135 bp
SV40 polyadenylation signal
HIV-1 Ψ
5982 .. 6107  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
5982 .. 6107  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
7346 .. 7463  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
7346 .. 7463  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
926 .. 1030  =  105 bp
AmpR promoter
926 .. 1030  =  105 bp
Rex NLS
9782 .. 9832  =  51 bp
17 amino acids  =  2.2 kDa
Product: nuclear localization signal from the human
T-cell leukemia virus type 1 (HTLV-1) Rex protein
Rex NLS
9782 .. 9832  =  51 bp
17 amino acids  =  2.2 kDa
Product: nuclear localization signal from the human
T-cell leukemia virus type 1 (HTLV-1) Rex protein
HA
8552 .. 8578  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
8552 .. 8578  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 ori
3076 .. 3211  =  136 bp
SV40 origin of replication
SV40 ori
3076 .. 3211  =  136 bp
SV40 origin of replication
SV40 ori
7595 .. 7730  =  136 bp
SV40 origin of replication
SV40 ori
7595 .. 7730  =  136 bp
SV40 origin of replication
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