pDONR222

Gateway® donor vector with attP1 and attP2 sites and a kanamycin resistance marker. The alternative pDONR™221 vector seems to be preferred.

Sequence Author: Thermo Fisher (Invitrogen)

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AcuI (4591) PspFI (4351) BseYI (4347) BssSI - BssSαI (4216) DrdI (4151) NspI (4047) PciI (4043) BspQI - SapI (3927) BglII (3715) PaeR7I - PspXI - XhoI (3680) NruI (3625) BspDI - ClaI (3589) EcoNI (3369) AsiSI - PvuI (3284) HindIII (3160) EcoRV (2788) BsaAI (2388) rrnB T2 terminator BsaHI (117) BbsI (227) HpaI (291) AflII (344) PspOMI (353) EcoO109I (354) ApaI (357) XmnI (842) PstI (957) BfuAI - BspMI (960) BsaI (1066) BstXI (1095) BmgBI (1174) BsaBI * (1195) SrfI (1208) BbvCI (1351) BstZ17I (1544) BssHII (1581) BamHI (1622) ScaI (1664) BtgI - NcoI - StyI (1776) BpmI (1961) EcoRI (2077) BspEI (2081) pDONR™222 4718 bp
AcuI  (4591)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
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).
PspFI  (4351)
1 site
C C C A G C G G G T C G
BseYI  (4347)
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.
BssSI  (4216)
1 site
C A C G A G G T G C T C
BssSαI  (4216)
1 site
C A C G A G G T G C T C
DrdI  (4151)
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.
NspI  (4047)
1 site
R C A T G Y Y G T A C R
PciI  (4043)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3927)
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  (3927)
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.
BglII  (3715)
1 site
A G A T C T T C T A G A
PaeR7I  (3680)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3680)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3680)
1 site
C T C G A G G A G C T C
NruI  (3625)
1 site
T C G C G A A G C G C T
BspDI  (3589)
1 site
A T C G A T T A G C T A
ClaI  (3589)
1 site
A T C G A T T A G C T A
EcoNI  (3369)
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.
AsiSI  (3284)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3284)
1 site
C G A T C G G C T A G C
HindIII  (3160)
1 site
A A G C T T T T C G A A
EcoRV  (2788)
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.
BsaAI  (2388)
1 site
Y A C G T R R T G C A Y
BsaHI  (117)
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  (227)
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  (291)
1 site
G T T A A C C A A T T G
AflII  (344)
1 site
C T T A A G G A A T T C
PspOMI  (353)
1 site
G G G C C C C C C G G G
EcoO109I  (354)
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  (357)
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  (842)
1 site
G A A N N N N T T C C T T N N N N A A G
PstI  (957)
1 site
C T G C A G G A C G T C
BfuAI  (960)
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  (960)
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  (1066)
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  (1095)
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  (1174)
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  (1195)
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.
SrfI  (1208)
1 site
G C C C G G G C C G G G C C C G
BbvCI  (1351)
1 site
C C T C A G C G G A G T C G
BstZ17I  (1544)
1 site
G T A T A C C A T A T G
BssHII  (1581)
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  (1622)
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  (1664)
1 site
A G T A C T T C A T G A
BtgI  (1776)
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  (1776)
1 site
C C A T G G G G T A C C
StyI  (1776)
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  (1961)
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  (2077)
1 site
G A A T T C C T T A A G
BspEI  (2081)
1 site
T C C G G A A G G C C T
KanR
2899 .. 3714  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
2899 .. 3714  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
CmR
1642 .. 2295  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1642 .. 2295  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4104 .. 4692  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4104 .. 4692  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ccdB
987 .. 1292  =  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
987 .. 1292  =  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.
attP1
360 .. 591  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™201 version)
attP1
360 .. 591  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™201 version)
attP2
2543 .. 2774  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™201 version)
attP2
2543 .. 2774  =  232 bp
recombination site for the Gateway® BP reaction (pDONR™201 version)
cat promoter
2296 .. 2398  =  103 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
cat promoter
2296 .. 2398  =  103 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
rrnB T1 terminator
177 .. 263  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
177 .. 263  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T2 terminator
58 .. 85  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
58 .. 85  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
M13 fwd
327 .. 343  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
327 .. 343  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
2816 .. 2832  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
2816 .. 2832  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  987 .. 1292  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  4438 .. 67  =  348 bp
ORF:  115 amino acids  =  12.9 kDa
ORF:  1612 .. 2295  =  684 bp
ORF:  227 amino acids  =  26.7 kDa
ORF:  2899 .. 3759  =  861 bp
ORF:  286 amino acids  =  32.8 kDa
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