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Plasmid Files

pAAV-Guide-it-Up Vector

CRISPR/Cas9 adeno-associated virus (AAV) vector for cloning and expression of a single guide RNA (sgRNA). Use together with pAAV‑Guide-it-Down Vector.

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pAAV-Guide-it-Up Vector Sequence and MappAAV-Guide-it-Up Vector.dna
Map and Sequence File   
Sequence Author:  Clontech
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 XmnI (5268) PsiI (4597) PmlI (3537) BspDI - ClaI (143) NdeI (383) SnaBI (489) AflII (733) Acc65I (781) KpnI (785) AarI (1166) AfeI (1717) PspOMI (2103) ApaI (2107) BstAPI (2283) BsrGI (2306) SphI (2349) BstEII (2421) BbsI (2662) EagI (3201) XcmI (3366) pAAV-Guide-it-Up Vector 7117 bp
XmnI  (5268)
1 site
G A A N N N N T T C C T T N N N N A A G
PsiI  (4597)
1 site
T T A T A A A A T A T T
PmlI  (3537)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BspDI  (143)
1 site
A T C G A T T A G C T A
ClaI  (143)
1 site
A T C G A T T A G C T A
NdeI  (383)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
SnaBI  (489)
1 site
T A C G T A A T G C A T
AflII  (733)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
Acc65I  (781)
1 site
G G T A C C C C A T G G
KpnI  (785)
1 site
G G T A C C C C A T G G
AarI  (1166)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
AfeI  (1717)
1 site
A G C G C T T C G C G A
PspOMI  (2103)
1 site
G G G C C C C C C G G G
ApaI  (2107)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BstAPI  (2283)
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.
BsrGI  (2306)
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.
SphI  (2349)
1 site
G C A T G C C G T A C G
BstEII  (2421)
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.
BbsI  (2662)
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.
EagI  (3201)
1 site
C G G C C G G C C G G C
XcmI  (3366)
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.
Cas9-Up
748 .. 3853  =  3106 bp
1035 amino acids  =  120.3 kDa
Cas9-Up
748 .. 3853  =  3106 bp
1035 amino acids  =  120.3 kDa
AmpR
5081 .. 5941  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5081 .. 5149  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5081 .. 5941  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5150 .. 5941  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5081 .. 5941  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
6112 .. 6700  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6112 .. 6700  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4239 .. 4694  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4239 .. 4694  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
210 .. 513  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
210 .. 513  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
514 .. 717  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
514 .. 717  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
AAV2 ITR
1 .. 141  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
1 .. 141  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
3860 .. 4000  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
3860 .. 4000  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
4976 .. 5080  =  105 bp
AmpR promoter
4976 .. 5080  =  105 bp
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