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pKANNIBAL

Vector for silencing genes in plants using intron-containing hairpin RNA (hpRNA).

 
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pKANNIBAL.dna
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NotI (6057) SpeI (6020) BmtI (6016) NheI (6012) SphI (6008) PstI - SbfI (6002) AhdI (5977) ApaI (5902) PspOMI (5898) BstEII (5847) BssHII (5753) MreI - SgrAI (5749) PasI (5678) AgeI (5623) BsmBI (5301) XbaI (5279) BamHI (5273) HindIII (5267) BspDI - ClaI (5262) PacI (5151) AanI (4582) AanI (4561) AanI (4544) KpnI (4466) Acc65I (4462) EcoRI (4456) PaeR7I - PspXI - XhoI (4450) BmgBI (4326) PshAI (4263) AccI (4053) HincII (4027) BsaBI (3935) StyI (3856) ScaI (3816) BsaI (3682) StuI (3664) MluI (3332) NotI (3097) SacI (3089) Eco53kI (3087) SfiI (3058) PflFI - Tth111I (5) PvuI (136) FspI (155) AanI (284) BsaAI - DraIII (412) BtgZI (413) AanI (1018) XcmI (1239) BtgI (1635) EcoRV (1767) BspQI - SapI (2806) MscI (3052) pKANNIBAL 6063 bp
NotI  (6057)
2 sites
G C G G C C G C C G C C G G C G
SpeI  (6020)
1 site
A C T A G T T G A T C A
BmtI  (6016)
1 site
G C T A G C C G A T C G
NheI  (6012)
1 site
G C T A G C C G A T C G
SphI  (6008)
1 site
G C A T G C C G T A C G
PstI  (6002)
1 site
C T G C A G G A C G T C
SbfI  (6002)
1 site
C C T G C A G G G G A C G T C C
AhdI  (5977)
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.
ApaI  (5902)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (5898)
1 site
G G G C C C C C C G G G
BstEII  (5847)
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.
BssHII  (5753)
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.
MreI  (5749)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (5749)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
PasI  (5678)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
AgeI  (5623)
1 site
A C C G G T T G G C C A
BsmBI  (5301)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
XbaI  (5279)
1 site
T C T A G A A G A T C T
BamHI  (5273)
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.
HindIII  (5267)
1 site
A A G C T T T T C G A A
BspDI  (5262)
1 site
A T C G A T T A G C T A
ClaI  (5262)
1 site
A T C G A T T A G C T A
PacI  (5151)
1 site
T T A A T T A A A A T T A A T T
AanI  (4582)
5 sites
T T A T A A A A T A T T
AanI  (4561)
5 sites
T T A T A A A A T A T T
AanI  (4544)
5 sites
T T A T A A A A T A T T
KpnI  (4466)
1 site
G G T A C C C C A T G G
Acc65I  (4462)
1 site
G G T A C C C C A T G G
EcoRI  (4456)
1 site
G A A T T C C T T A A G
PaeR7I  (4450)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4450)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4450)
1 site
C T C G A G G A G C T C
BmgBI  (4326)
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.
PshAI  (4263)
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.
AccI  (4053)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
HincII  (4027)
1 site
G T Y R A C C A R Y T G
BsaBI  (3935)
1 site
G A T N N N N A T C C T A N N N N T A G
StyI  (3856)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
ScaI  (3816)
1 site
A G T A C T T C A T G A
BsaI  (3682)
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.
StuI  (3664)
1 site
A G G C C T T C C G G A
MluI  (3332)
1 site
A C G C G T T G C G C A
NotI  (3097)
2 sites
G C G G C C G C C G C C G G C G
SacI  (3089)
1 site
G A G C T C C T C G A G
Eco53kI  (3087)
1 site
G A G C T C C T C G A G
SfiI  (3058)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
PflFI  (5)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (5)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PvuI  (136)
1 site
C G A T C G G C T A G C
FspI  (155)
1 site
T G C G C A A C G C G T
AanI  (284)
5 sites
T T A T A A A A T A T T
BsaAI  (412)
1 site
Y A C G T R R T G C A Y
DraIII  (412)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BtgZI  (413)
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.
AanI  (1018)
5 sites
T T A T A A A A T A T T
XcmI  (1239)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BtgI  (1635)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
EcoRV  (1767)
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.
BspQI  (2806)
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  (2806)
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.
MscI  (3052)
1 site
T G G C C A A C C G G T
KanR
1075 .. 1869  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
1075 .. 1869  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
PDK intron
4475 .. 5241  =  767 bp
pyruvate orthophosphate dikinase intron from Flaveria trinervia
PDK intron
4475 .. 5241  =  767 bp
pyruvate orthophosphate dikinase intron from Flaveria trinervia
OCS terminator
5288 .. 5994  =  707 bp
octopine synthase terminator
OCS terminator
5288 .. 5994  =  707 bp
octopine synthase terminator
ori
2045 .. 2633  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2045 .. 2633  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
215 .. 643  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
215 .. 643  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CaMV 35S promoter
4102 .. 4447  =  346 bp
strong constitutive promoter from cauliflower mosaic virus
CaMV 35S promoter
4102 .. 4447  =  346 bp
strong constitutive promoter from cauliflower mosaic virus
AmpR promoter
670 .. 774  =  105 bp
AmpR promoter
670 .. 774  =  105 bp
MCS 2
5261 .. 5284  =  24 bp
multiple cloning site 2
MCS 2
5261 .. 5284  =  24 bp
multiple cloning site 2
MCS 1
4449 .. 4467  =  19 bp
multiple cloning site 1
MCS 1
4449 .. 4467  =  19 bp
multiple cloning site 1
ORF:  775 .. 999  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  1075 .. 1869  =  795 bp
ORF:  264 amino acids  =  31.0 kDa
ORF:  3031 .. 3930  =  900 bp
ORF:  299 amino acids  =  33.5 kDa
ORF:  3605 .. 3868  =  264 bp
ORF:  87 amino acids  =  10.4 kDa
ORF:  3920 .. 4348  =  429 bp
ORF:  142 amino acids  =  15.9 kDa
ORF:  3553 .. 3921  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  1506 .. 1736  =  231 bp
ORF:  76 amino acids  =  8.3 kDa
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