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Plasmid Files

pEarleyGate 201

Gateway®-compatible plant transformation vector with an N-terminal HA tag.

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pEarleyGate 201 Sequence and MappEarleyGate 201.dna
Map and Sequence File   
Sequence Author:  Pikaard Lab
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 LB T-DNA repeat BclI * (11,542) PsiI (11,411) BspHI (11,085) EcoRV (10,662) PluTI (9403) SfoI (9401) NarI (9400) KasI (9399) EcoNI (8927) BmtI (8618) NheI (8614) BsiWI (8498) PmeI (5486) AseI (92) Acc65I (407) KpnI (411) FspAI (521) AarI (585) AfeI (758) BstBI (1770) StuI (1868) PshAI (2467) PaeR7I - PspXI - XhoI (2654) TMV Ω ATG HA attR1 lac UV5 promoter BspEI (3212) NcoI (3517) BbvCI (3941) SrfI (4087) BstXI (4204) AvrII (4476) PacI (4522) SpeI (4534) MfeI (4838) BstEII (5110) AhdI (5240) SbfI (5265) HindIII (5273) PvuI (5399) pEarleyGate 201 11,779 bp
BclI  (11,542)
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  (11,411)
1 site
T T A T A A A A T A T T
BspHI  (11,085)
1 site
T C A T G A A G T A C T
EcoRV  (10,662)
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.
PluTI  (9403)
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.
SfoI  (9401)
1 site
G G C G C C C C G C G G
NarI  (9400)
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.
KasI  (9399)
1 site
G G C G C C C C G C G G
EcoNI  (8927)
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.
BmtI  (8618)
1 site
G C T A G C C G A T C G
NheI  (8614)
1 site
G C T A G C C G A T C G
BsiWI  (8498)
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  (5486)
1 site
G T T T A A A C C A A A T T T G
AseI  (92)
1 site
A T T A A T T A A T T A
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
AarI  (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 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.
AfeI  (758)
1 site
A G C G C T T C G C G A
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.
PspXI  (2654)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2654)
1 site
C T C G A G G A G C T C
BspEI  (3212)
1 site
T C C G G A A G G C C T
NcoI  (3517)
1 site
C C A T G G G G T A C C
BbvCI  (3941)
1 site
C C T C A G C G G A G T C G
SrfI  (4087)
1 site
G C C C G G G C C G G G C C C G
BstXI  (4204)
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  (4476)
1 site
C C T A G G G G A T C C
PacI  (4522)
1 site
T T A A T T A A A A T T A A T T
SpeI  (4534)
1 site
A C T A G T T G A T C A
MfeI  (4838)
1 site
C A A T T G G T T A A C
BstEII  (5110)
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  (5240)
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  (5265)
1 site
C C T G C A G G G G A C G T C C
HindIII  (5273)
1 site
A A G C T T T T C G A A
PvuI  (5399)
1 site
C G A T C G G C T A G C
pVS1 RepA
7883 .. 8956  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from Pseudomonas
plasmid pVS1
pVS1 RepA
7883 .. 8956  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from Pseudomonas
plasmid pVS1
KanR
10,561 .. 11,355  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
10,561 .. 11,355  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
OCS terminator
4550 .. 5257  =  708 bp
octopine synthase terminator
OCS terminator
4550 .. 5257  =  708 bp
octopine synthase terminator
CmR
3003 .. 3662  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
3003 .. 3662  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
pVS1 StaA
6825 .. 7454  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from Pseudomonas
plasmid pVS1
pVS1 StaA
6825 .. 7454  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from Pseudomonas
plasmid pVS1
ori
9886 .. 10,474  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9886 .. 10,474  =  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
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
4004 .. 4309  =  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
4004 .. 4309  =  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
9022 .. 9216  =  195 bp
origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)
pVS1 oriV
9022 .. 9216  =  195 bp
origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)
bom
9560 .. 9700  =  141 bp
basis of mobility region from pBR322
bom
9560 .. 9700  =  141 bp
basis of mobility region from pBR322
attR2
4350 .. 4474  =  125 bp
recombination site for the Gateway® LR reaction
attR2
4350 .. 4474  =  125 bp
recombination site for the Gateway® LR reaction
attR1
2770 .. 2889  =  120 bp
recombination site for the Gateway® LR reaction
attR1
2770 .. 2889  =  120 bp
recombination site for the Gateway® LR reaction
TMV Ω
2681 .. 2735  =  55 bp
translational enhancer from the tobacco mosaic
virus 5'-leader sequence (Gallie et al., 1988)
TMV Ω
2681 .. 2735  =  55 bp
translational enhancer from the tobacco mosaic
virus 5'-leader sequence (Gallie et al., 1988)
lac UV5 promoter
2919 .. 2949  =  31 bp
   Segment 1:  -35  
   2919 .. 2924  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2919 .. 2949  =  31 bp
   Segment 2:  
   2925 .. 2942  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2919 .. 2949  =  31 bp
   Segment 3:  -10  
   2943 .. 2949  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2919 .. 2949  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
HA
2740 .. 2766  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
2740 .. 2766  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
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
5501 .. 5525  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
5501 .. 5525  =  25 bp
right border repeat from nopaline C58 T-DNA
ATG
2737 .. 2739  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2737 .. 2739  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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