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Plasmid Files

pDONR™221 P5-P4

Gateway® donor vector with attP5 and attP4 sites and a kanamycin resistance marker.

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pDONR221 P5-P4 Sequence and MappDONR221 P5-P4.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PciI (4720) DrdI (4618) BssS α I (4547) PspFI (4420) BseYI (4416) AcuI (4178) AsiSI - PvuI (3545) EcoNI (3457) NruI (3202) EcoRV (2963) EagI - NotI (2954) BsaAI (2550) BspEI (2243) NspI (4724) BspQI - SapI (111) BsaHI (327) BbsI (437) HpaI (501) M13 fwd AflII (554) PspOMI (563) EcoO109I (564) ApaI (567) XmnI (1004) BfuAI - BspMI (1122) BsaI (1228) BstXI (1257) BmgBI (1336) BsaBI * (1357) TspMI - XmaI (1368) SmaI - SrfI (1370) BsrGI (1393) BbvCI (1513) BssHII (1743) BamHI (1784) ScaI (1826) BtgI - NcoI - StyI (1938) BpmI (2123) EcoRI (2239) pDONR™221 P5-P4 4726 bp
PciI  (4720)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (4618)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BssSαI  (4547)
1 site
C A C G A G G T G C T C
PspFI  (4420)
1 site
C C C A G C G G G T C G
BseYI  (4416)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
AcuI  (4178)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
AsiSI  (3545)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3545)
1 site
C G A T C G G C T A G C
EcoNI  (3457)
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.
NruI  (3202)
1 site
T C G C G A A G C G C T
EcoRV  (2963)
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.
EagI  (2954)
1 site
C G G C C G G C C G G C
NotI  (2954)
1 site
G C G G C C G C C G C C G G C G
BsaAI  (2550)
1 site
Y A C G T R R T G C A Y
BspEI  (2243)
1 site
T C C G G A A G G C C T
NspI  (4724)
1 site
R C A T G Y Y G T A C R
BspQI  (111)
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  (111)
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.
BsaHI  (327)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
BbsI  (437)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
HpaI  (501)
1 site
G T T A A C C A A T T G
AflII  (554)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
PspOMI  (563)
1 site
G G G C C C C C C G G G
EcoO109I  (564)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
ApaI  (567)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
XmnI  (1004)
1 site
G A A N N N N T T C C T T N N N N A A G
BfuAI  (1122)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1122)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsaI  (1228)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BstXI  (1257)
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.
BmgBI  (1336)
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.
BsaBI  (1357)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
TspMI  (1368)
1 site
C C C G G G G G G C C C
XmaI  (1368)
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  (1370)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (1370)
1 site
G C C C G G G C C G G G C C C G
BsrGI  (1393)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BbvCI  (1513)
1 site
C C T C A G C G G A G T C G
BssHII  (1743)
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.
BamHI  (1784)
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.
ScaI  (1826)
1 site
A G T A C T T C A T G A
BtgI  (1938)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (1938)
1 site
C C A T G G G G T A C C
StyI  (1938)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BpmI  (2123)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
EcoRI  (2239)
1 site
G A A T T C C T T A A G
KanR
3120 .. 3929  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
3120 .. 3929  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
CmR
1804 .. 2457  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1804 .. 2457  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4076 .. 4664  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4076 .. 4664  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
1149 .. 1454  =  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
1149 .. 1454  =  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.
attP5
570 .. 801  =  232 bp
recombination site for the Gateway® BP reaction
attP5
570 .. 801  =  232 bp
recombination site for the Gateway® BP reaction
attP4
2705 .. 2936  =  232 bp
recombination site for the Gateway® BP reaction
attP4
2705 .. 2936  =  232 bp
recombination site for the Gateway® BP reaction
cat promoter
2458 .. 2560  =  103 bp
promoter of the E. coli cat gene
cat promoter
2458 .. 2560  =  103 bp
promoter of the E. coli cat gene
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
M13 fwd
537 .. 553  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
537 .. 553  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2991 .. 3007  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2991 .. 3007  =  17 bp
common sequencing primer, one of multiple similar
variants
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