pEarleyGate 205

Gateway®-compatible plant transformation vector with a C-terminal TAP tag.

Sequence Author: Pikaard Lab

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BclI * (11,951) PsiI (11,820) BspHI (11,494) EcoRV (11,071) EcoNI (9336) BsiWI (8907) PmeI (5895) LB T-DNA repeat Acc65I (407) KpnI (411) FspAI (521) PaqCI (585) BstBI (1770) StuI (1868) PshAI (2467) PaeR7I - XhoI (2654) attR1 lac UV5 promoter BspEI (3105) NcoI (3410) BbvCI (3834) SrfI (3980) BstXI (4097) attR2 AvrII (4369) TEV Site ProtA ProtA PacI (4931) SpeI (4943) MfeI (5247) BstEII (5519) AhdI (5649) SbfI (5674) HindIII (5682) PvuI (5808) pEarleyGate 205 12,188 bp
BclI  (11,951)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
PsiI  (11,820)
1 site
T T A T A A A A T A T T
BspHI  (11,494)
1 site
T C A T G A A G T A C T
EcoRV  (11,071)
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.
EcoNI  (9336)
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.
BsiWI  (8907)
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.
PmeI  (5895)
1 site
G T T T A A A C C A A A T T T G
Acc65I  (407)
1 site
G G T A C C C C A T G G
KpnI  (411)
1 site
G G T A C C C C A T G G
FspAI  (521)
1 site
R T G C G C A Y Y A C G C G T R
PaqCI  (585)
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.
BstBI  (1770)
1 site
T T C G A A A A G C T T
StuI  (1868)
1 site
A G G C C T T C C G G A
PshAI  (2467)
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.
PaeR7I  (2654)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2654)
1 site
C T C G A G G A G C T C
BspEI  (3105)
1 site
T C C G G A A G G C C T
NcoI  (3410)
1 site
C C A T G G G G T A C C
BbvCI  (3834)
1 site
C C T C A G C G G A G T C G
SrfI  (3980)
1 site
G C C C G G G C C G G G C C C G
BstXI  (4097)
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.
AvrII  (4369)
1 site
C C T A G G G G A T C C
PacI  (4931)
1 site
T T A A T T A A A A T T A A T T
SpeI  (4943)
1 site
A C T A G T T G A T C A
MfeI  (5247)
1 site
C A A T T G G T T A A C
BstEII  (5519)
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.
AhdI  (5649)
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.
SbfI  (5674)
1 site
C C T G C A G G G G A C G T C C
HindIII  (5682)
1 site
A A G C T T T T C G A A
PvuI  (5808)
1 site
C G A T C G G C T A G C
pVS1 RepA
8292 .. 9365  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from Pseudomonas plasmid pVS1
pVS1 RepA
8292 .. 9365  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from Pseudomonas plasmid pVS1
KanR
10,970 .. 11,764  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
10,970 .. 11,764  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
OCS terminator
4959 .. 5666  =  708 bp
octopine synthase terminator
OCS terminator
4959 .. 5666  =  708 bp
octopine synthase terminator
CmR
2896 .. 3555  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2896 .. 3555  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
pVS1 StaA
7234 .. 7863  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from Pseudomonas plasmid pVS1
pVS1 StaA
7234 .. 7863  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from Pseudomonas plasmid pVS1
ori
10,295 .. 10,883  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
10,295 .. 10,883  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
BlpR
373 .. 924  =  552 bp
183 amino acids  =  20.6 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialophos or phosphinothricin
BlpR
373 .. 924  =  552 bp
183 amino acids  =  20.6 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialophos or phosphinothricin
CBP
4381 .. 4458  =  78 bp
26 amino acids  =  3.0 kDa
Product: calmodulin-binding peptide
derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium
CBP
4381 .. 4458  =  78 bp
26 amino acids  =  3.0 kDa
Product: calmodulin-binding peptide
derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium
TEV Site
4486 .. 4506  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) recognition and cleavage site
TEV Site
4486 .. 4506  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) recognition and cleavage site
ProtA
4546 .. 4719  =  174 bp
58 amino acids  =  6.6 kDa
Product: IgG-binding unit of Staphylococcus aureus protein A
ProtA
4546 .. 4719  =  174 bp
58 amino acids  =  6.6 kDa
Product: IgG-binding unit of Staphylococcus aureus protein A
ProtA
4720 .. 4893  =  174 bp
58 amino acids  =  6.6 kDa
Product: IgG-binding unit of Staphylococcus aureus protein A
ProtA
4720 .. 4893  =  174 bp
58 amino acids  =  6.6 kDa
Product: IgG-binding unit of Staphylococcus aureus protein A
MAS promoter
930 .. 1310  =  381 bp
mannopine synthase promoter (Velten et al., 1984)
MAS promoter
930 .. 1310  =  381 bp
mannopine synthase promoter (Velten et al., 1984)
CaMV 35S promoter
2306 .. 2651  =  346 bp
strong constitutive promoter from cauliflower mosaic virus
CaMV 35S promoter
2306 .. 2651  =  346 bp
strong constitutive promoter from cauliflower mosaic virus
ccdB
3897 .. 4202  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
ccdB
3897 .. 4202  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
MAS terminator
111 .. 363  =  253 bp
mannopine synthase terminator
MAS terminator
111 .. 363  =  253 bp
mannopine synthase terminator
pVS1 oriV
9431 .. 9625  =  195 bp
origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
pVS1 oriV
9431 .. 9625  =  195 bp
origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
bom
9969 .. 10,109  =  141 bp
basis of mobility region from pBR322
bom
9969 .. 10,109  =  141 bp
basis of mobility region from pBR322
attR2
4243 .. 4367  =  125 bp
recombination site for the Gateway® LR reaction
attR2
4243 .. 4367  =  125 bp
recombination site for the Gateway® LR reaction
attR1
2663 .. 2782  =  120 bp
recombination site for the Gateway® LR reaction
attR1
2663 .. 2782  =  120 bp
recombination site for the Gateway® LR reaction
lac UV5 promoter
2812 .. 2842  =  31 bp
3 segments
   Segment 1:  -35  
   2812 .. 2817  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2812 .. 2842  =  31 bp
3 segments
   Segment 2:  
   2818 .. 2835  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2812 .. 2842  =  31 bp
3 segments
   Segment 3:  -10  
   2836 .. 2842  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2812 .. 2842  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
LB T-DNA repeat
1 .. 25  =  25 bp
left border repeat from nopaline C58 T-DNA
LB T-DNA repeat
1 .. 25  =  25 bp
left border repeat from nopaline C58 T-DNA
RB T-DNA repeat
5910 .. 5934  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
5910 .. 5934  =  25 bp
right border repeat from nopaline C58 T-DNA
ORF:  448 .. 957  =  510 bp
ORF:  169 amino acids  =  18.0 kDa
ORF:  2896 .. 3555  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  4375 .. 4926  =  552 bp
ORF:  183 amino acids  =  20.5 kDa
ORF:  7234 .. 7863  =  630 bp
ORF:  209 amino acids  =  22.1 kDa
ORF:  275 .. 607  =  333 bp
ORF:  110 amino acids  =  11.9 kDa
ORF:  1730 .. 2134  =  405 bp
ORF:  134 amino acids  =  15.5 kDa
ORF:  5546 .. 5836  =  291 bp
ORF:  96 amino acids  =  10.1 kDa
ORF:  1809 .. 2072  =  264 bp
ORF:  87 amino acids  =  10.4 kDa
ORF:  2124 .. 2552  =  429 bp
ORF:  142 amino acids  =  15.9 kDa
ORF:  3897 .. 4202  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  6249 .. 6935  =  687 bp
ORF:  228 amino acids  =  25.1 kDa
ORF:  8190 .. 9365  =  1176 bp
ORF:  391 amino acids  =  43.9 kDa
ORF:  11,103 .. 11,333  =  231 bp
ORF:  76 amino acids  =  8.3 kDa
ORF:  6972 .. 7286  =  315 bp
ORF:  104 amino acids  =  12.0 kDa
ORF:  8160 .. 8732  =  573 bp
ORF:  190 amino acids  =  20.6 kDa
ORF:  1757 .. 2125  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  6281 .. 6631  =  351 bp
ORF:  116 amino acids  =  12.6 kDa
ORF:  8138 .. 8395  =  258 bp
ORF:  85 amino acids  =  9.6 kDa
ORF:  10,970 .. 11,764  =  795 bp
ORF:  264 amino acids  =  31.0 kDa
ORF:  12,009 .. 321  =  501 bp
ORF:  166 amino acids  =  18.5 kDa
ORF:  373 .. 924  =  552 bp
ORF:  183 amino acids  =  20.6 kDa
ORF:  6700 .. 6972  =  273 bp
ORF:  90 amino acids  =  9.9 kDa
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