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Plasmid Files

pGADT7-Rec

High-copy yeast vector for fusing a cDNA to the GAL4 activation domain by in vivo recombination.

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pGADT7-Rec Sequence and MappGADT7-Rec.dna
Map and Sequence File   
Sequence Author:  Clontech
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 NsiI (7769) BsaAI - SnaBI (7413) BmgBI (6971) AatII (6639) ZraI (6637) PvuI (6087) BmrI (5757) AhdI (5717) AlwNI (5240) BspQI - SapI (4708) BspEI * (4597) NotI (4588) BmtI (4570) NheI (4566) SbfI (12) KasI (144) NarI (145) SfoI (146) PluTI (148) NgoMIV (210) NaeI (212) Bpu10I (291) BsmI (325) BtgZI (351) PacI (685) BsaBI (1009) ADH1 promoter BsgI (1421) ATG SV40 NLS Acc65I (1548) KpnI (1552) RsrII (1626) SexAI * (1766) MluI (1795) BglII (1897) T7 promoter ATG HA NdeI (1969) EcoRI (1989) TspMI - XmaI (2034) SmaI (2036) CDS III XbaI (2065) BspDI - ClaI (2080) BamHI (2088) Eco53kI (2099) PaeR7I - PspXI - XhoI (2100) BanII - SacI (2101) AflII (3565) BstEII (3845) PpuMI (3990) pGADT7-Rec 8058 bp
NsiI  (7769)
1 site
A T G C A T T A C G T A
BsaAI  (7413)
1 site
Y A C G T R R T G C A Y
SnaBI  (7413)
1 site
T A C G T A A T G C A T
BmgBI  (6971)
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.
AatII  (6639)
1 site
G A C G T C C T G C A G
ZraI  (6637)
1 site
G A C G T C C T G C A G
PvuI  (6087)
1 site
C G A T C G G C T A G C
BmrI  (5757)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the
absence of magnesium.
AhdI  (5717)
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.
AlwNI  (5240)
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.
BspQI  (4708)
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  (4708)
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.
BspEI  (4597)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
NotI  (4588)
1 site
G C G G C C G C C G C C G G C G
BmtI  (4570)
1 site
G C T A G C C G A T C G
NheI  (4566)
1 site
G C T A G C C G A T C G
SbfI  (12)
1 site
C C T G C A G G G G A C G T C C
KasI  (144)
1 site
G G C G C C C C G C G G
NarI  (145)
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  (146)
1 site
G G C G C C C C G C G G
PluTI  (148)
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.
NgoMIV  (210)
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  (212)
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.
Bpu10I  (291)
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.
BsmI  (325)
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.
BtgZI  (351)
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.
PacI  (685)
1 site
T T A A T T A A A A T T A A T T
BsaBI  (1009)
1 site
G A T N N N N A T C C T A N N N N T A G
BsgI  (1421)
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).
Acc65I  (1548)
1 site
G G T A C C C C A T G G
KpnI  (1552)
1 site
G G T A C C C C A T G G
RsrII  (1626)
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.
SexAI  (1766)
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.
MluI  (1795)
1 site
A C G C G T T G C G C A
BglII  (1897)
1 site
A G A T C T T C T A G A
NdeI  (1969)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
EcoRI  (1989)
1 site
G A A T T C C T T A A G
TspMI  (2034)
1 site
C C C G G G G G G C C C
XmaI  (2034)
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.
SmaI  (2036)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
XbaI  (2065)
1 site
T C T A G A A G A T C T
BspDI  (2080)
1 site
A T C G A T T A G C T A
ClaI  (2080)
1 site
A T C G A T T A G C T A
BamHI  (2088)
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.
Eco53kI  (2099)
1 site
G A G C T C C T C G A G
PaeR7I  (2100)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2100)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2100)
1 site
C T C G A G G A G C T C
BanII  (2101)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (2101)
1 site
G A G C T C C T C G A G
AflII  (3565)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BstEII  (3845)
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.
PpuMI  (3990)
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.
2μ ori
6891 .. 8055  =  1165 bp
yeast 2μ plasmid origin of replication
2μ ori
6891 .. 8055  =  1165 bp
yeast 2μ plasmid origin of replication
LEU2
2790 .. 3884  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
LEU2
2790 .. 3884  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
AmpR
5644 .. 6504  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5644 .. 6435  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5644 .. 6504  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6436 .. 6504  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5644 .. 6504  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ADH1 promoter
771 .. 1475  =  705 bp
promoter for alcohol dehydrogenase 1
ADH1 promoter
771 .. 1475  =  705 bp
promoter for alcohol dehydrogenase 1
ori
4885 .. 5473  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4885 .. 5473  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
LEU2 promoter
3885 .. 4290  =  406 bp
LEU2 promoter
3885 .. 4290  =  406 bp
GAL4 activation domain
1557 .. 1898  =  342 bp
114 amino acids  =  12.4 kDa
Product: activation domain of the GAL4
transcriptional activator
GAL4 activation domain
1557 .. 1898  =  342 bp
114 amino acids  =  12.4 kDa
Product: activation domain of the GAL4
transcriptional activator
ADH1 terminator
2486 .. 2673  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
2486 .. 2673  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
6505 .. 6609  =  105 bp
AmpR promoter
6505 .. 6609  =  105 bp
SMART III
2000 .. 2035  =  36 bp
matches the SMART III Oligonucleotide for cDNA
synthesis
SMART III
2000 .. 2035  =  36 bp
matches the SMART III Oligonucleotide for cDNA
synthesis
HA
1941 .. 1967  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
1941 .. 1967  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
CDS III
2046 .. 2070  =  25 bp
matches the CDS III Primer for cDNA synthesis
CDS III
2046 .. 2070  =  25 bp
matches the CDS III Primer for cDNA synthesis
SV40 NLS
1521 .. 1541  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1521 .. 1541  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
1904 .. 1922  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1904 .. 1922  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ATG
1491 .. 1493  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1491 .. 1493  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1935 .. 1937  =  3 bp
1 amino acid  =  149.2 Da
Product: in vitro start codon
ATG
1935 .. 1937  =  3 bp
1 amino acid  =  149.2 Da
Product: in vitro start codon
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