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Plasmid Files

pUC119-gRNA

CRISPR plasmid for expressing a single guide RNA (sgRNA) in plant cells. Use together with HBT‑pcoCas9.

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pUC119-gRNA Sequence and MappUC119-gRNA.dna
Map and Sequence File   
Sequence Author:  Sheen Lab / Addgene #52255
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 BspQI - SapI (3763) AflIII - PciI (3646) AlwNI (3237) AhdI (2758) BsaI (2692) BpmI (2689) NmeAIII (2611) ScaI (2277) AatII (1839) HindIII (234) SalI (246) AccI (247) HincII (248) TspMI - XmaI (253) SmaI - SrfI (255) Acc65I (260) KpnI (264) AsiSI (270) NruI (276) PstI - SbfI (285) BfuAI - BspMI (288) PI-PspI (335) Bsu36I (356) I-CeuI (360) I-SceI (387) PacI (397) Eco53kI (403) SacI (405) AfeI (655) polIII terminator BsrGI (830) SwaI (871) BamHI (876) SnaBI (884) PaeR7I - PspXI - XhoI (888) EcoRI (894) KasI (1055) NarI (1056) SfoI (1057) PluTI (1059) NgoMIV (1254) NaeI (1256) DraIII (1362) PsiI (1487) PfoI (1719) EcoO109I (1778) ZraI (1837) pUC119-gRNA 3771 bp
BspQI  (3763)
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  (3763)
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.
AflIII  (3646)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (3646)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AlwNI  (3237)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
AhdI  (2758)
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.
BsaI  (2692)
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.
BpmI  (2689)
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.
NmeAIII  (2611)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
ScaI  (2277)
1 site
A G T A C T T C A T G A
AatII  (1839)
1 site
G A C G T C C T G C A G
HindIII  (234)
1 site
A A G C T T T T C G A A
SalI  (246)
1 site
G T C G A C C A G C T G
AccI  (247)
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  (248)
1 site
G T Y R A C C A R Y T G
TspMI  (253)
1 site
C C C G G G G G G C C C
XmaI  (253)
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  (255)
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  (255)
1 site
G C C C G G G C C G G G C C C G
Acc65I  (260)
1 site
G G T A C C C C A T G G
KpnI  (264)
1 site
G G T A C C C C A T G G
AsiSI  (270)
1 site
G C G A T C G C C G C T A G C G
NruI  (276)
1 site
T C G C G A A G C G C T
PstI  (285)
1 site
C T G C A G G A C G T C
SbfI  (285)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (288)
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  (288)
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.
PI-PspI  (335)
1 site
T G G C A A A C A G C T A T T A T G G G T A T T A T G G G T A C C G T T T G T C G A T A A T A C C C A T A A T A C C C A

PI-PspI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
Bsu36I  (356)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
I-CeuI  (360)
1 site
T A A C T A T A A C G G T C C T A A G G T A G C G A A T T G A T A T T G C C A G G A T T C C A T C G C T

I-CeuI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
I-SceI  (387)
1 site
T A G G G A T A A C A G G G T A A T A T C C C T A T T G T C C C A T T A

I-SceI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
After cleavage, I-SceI can remain bound to DNA and alter its
electrophoretic mobility.
PacI  (397)
1 site
T T A A T T A A A A T T A A T T
Eco53kI  (403)
1 site
G A G C T C C T C G A G
SacI  (405)
1 site
G A G C T C C T C G A G
AfeI  (655)
1 site
A G C G C T T C G C G A
BsrGI  (830)
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.
SwaI  (871)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BamHI  (876)
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.
SnaBI  (884)
1 site
T A C G T A A T G C A T
PaeR7I  (888)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (888)
1 site
V C T C G A G B B G A G C T C V
XhoI  (888)
1 site
C T C G A G G A G C T C
EcoRI  (894)
1 site
G A A T T C C T T A A G
KasI  (1055)
1 site
G G C G C C C C G C G G
NarI  (1056)
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.
SfoI  (1057)
1 site
G G C G C C C C G C G G
PluTI  (1059)
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.
NgoMIV  (1254)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (1256)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
DraIII  (1362)
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.
PsiI  (1487)
1 site
T T A T A A A A T A T T
PfoI  (1719)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
EcoO109I  (1778)
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.
ZraI  (1837)
1 site
G A C G T C C T G C A G
AmpR
1971 .. 2831  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1971 .. 2039  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1971 .. 2831  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2040 .. 2831  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1971 .. 2831  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3002 .. 3590  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3002 .. 3590  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1129 .. 1584  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1129 .. 1584  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
AtU6-1 promoter
407 .. 710  =  304 bp
promoter for the Arabidopsis thaliana U6-1 snRNA
gene (Waibel and Filipowicz, 1990)
AtU6-1 promoter
407 .. 710  =  304 bp
promoter for the Arabidopsis thaliana U6-1 snRNA
gene (Waibel and Filipowicz, 1990)
AmpR promoter
1866 .. 1970  =  105 bp
AmpR promoter
1866 .. 1970  =  105 bp
gRNA scaffold
731 .. 806  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
731 .. 806  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
polIII terminator
807 .. 812  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
807 .. 812  =  6 bp
RNA polymerase III transcription terminator
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