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Plasmid Files

pGuide-it-sgRNA1 Vector (Linear)

Linearized CRISPR/Cas9 vector with an improved scaffold, for cloning and expression of a single guide RNA (sgRNA).

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pGuide-it-sgRNA1 Vector (Linear).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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End (3020) Guide-it™ Sequencing Primer 1 (2943 .. 2966) BsaAI (2885) BglII (2761) Bsu36I (2743) ApoI - EcoRI (2732) PluTI (2575) SfoI (2573) NarI (2572) KasI (2571) BstAPI (2521) PfoI (2382) AatII (2271) ZraI (2269) SspI (2153) XmnI (1948) ScaI (1829) TsoI (1748) NmeAIII (1497) Start (0) HindIII (97) BspQI - SapI (340) AflIII - PciI (456) BseYI (760) PspFI (764) AlwNI (872) AhdI (1349) BsaI (1410) BpmI (1419) BsrFI (1429) pGuide-it-sgRNA1 Vector (Linear) 3020 bp
End  (3020)
0 sites
BsaAI  (2885)
1 site
Y A C G T R R T G C A Y
BglII  (2761)
1 site
A G A T C T T C T A G A
Bsu36I  (2743)
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.
ApoI  (2732)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (2732)
1 site
G A A T T C C T T A A G
PluTI  (2575)
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  (2573)
1 site
G G C G C C C C G C G G
NarI  (2572)
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  (2571)
1 site
G G C G C C C C G C G G
BstAPI  (2521)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
PfoI  (2382)
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.
AatII  (2271)
1 site
G A C G T C C T G C A G
ZraI  (2269)
1 site
G A C G T C C T G C A G
SspI  (2153)
1 site
A A T A T T T T A T A A
XmnI  (1948)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (1829)
1 site
A G T A C T T C A T G A
TsoI  (1748)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
NmeAIII  (1497)
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).
Start  (0)
0 sites
HindIII  (97)
1 site
A A G C T T T T C G A A
BspQI  (340)
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  (340)
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  (456)
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  (456)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (760)
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.
PspFI  (764)
1 site
C C C A G C G G G T C G
AlwNI  (872)
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  (1349)
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  (1410)
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  (1419)
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.
BsrFI  (1429)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
Guide-it™ Sequencing Primer 1
24-mer  /  38% GC
1 binding site
2943 .. 2966  =  24 annealed bases
Tm  =  56°C
AmpR
1276 .. 2136  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1276 .. 2067  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1276 .. 2136  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2068 .. 2136  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1276 .. 2136  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
517 .. 1105  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
517 .. 1105  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
U6 promoter
2776 .. 3016  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
2776 .. 3016  =  241 bp
RNA polymerase III promoter for human U6 snRNA
AmpR promoter
2137 .. 2241  =  105 bp
AmpR promoter
2137 .. 2241  =  105 bp
gRNA scaffold
1 .. 86  =  86 bp
improved guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
1 .. 86  =  86 bp
improved guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
ORF:  1406 .. 1672  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2522 .. 2746  =  225 bp
ORF:  74 amino acids  =  8.8 kDa
ORF:  1276 .. 2136  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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