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Plasmid Files

pAAV-Guide-it-Down Vector

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

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pAAV-Guide-it-Down Vector (Linear) Sequence and MappAAV-Guide-it-Down Vector (Linear).dna
Map and Sequence File   
Sequence Author:  Clontech
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 End (6306) NdeI (6238) Guide-it™ Sequencing Primer 1 (6227 .. 6250) NsiI (5779) BbvCI (5744) AleI (5518) BamHI (5453) AccI (5424) SV40 NLS BspEI (5396) AgeI (5342) AfeI (5288) MreI - SgrAI (4934) KflI (4663) PmlI (4075) SacII (3877) BtgZI (3439) HindIII (3278) Start (0) polIII terminator BglII (147) ZraI (526) AatII (528) TsoI (1049) DrdI (2233) EcoRV (2770) BstAPI (2821) BsrGI (2844) BstEII (2959) pAAV-Guide-it-Down Vector 6306 bp
End  (6306)
0 sites
NdeI  (6238)
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.
NsiI  (5779)
1 site
A T G C A T T A C G T A
BbvCI  (5744)
1 site
C C T C A G C G G A G T C G
AleI  (5518)
1 site
C A C N N N N G T G G T G N N N N C A C
BamHI  (5453)
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.
AccI  (5424)
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.
BspEI  (5396)
1 site
T C C G G A A G G C C T
AgeI  (5342)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
AfeI  (5288)
1 site
A G C G C T T C G C G A
MreI  (4934)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (4934)
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.
KflI  (4663)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PmlI  (4075)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
SacII  (3877)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BtgZI  (3439)
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.
HindIII  (3278)
1 site
A A G C T T T T C G A A
Start  (0)
0 sites
BglII  (147)
1 site
A G A T C T T C T A G A
ZraI  (526)
1 site
G A C G T C C T G C A G
AatII  (528)
1 site
G A C G T C C T G C A G
TsoI  (1049)
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).
DrdI  (2233)
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.
EcoRV  (2770)
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.
BstAPI  (2821)
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  (2844)
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.
BstEII  (2959)
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.
Guide-it™ Sequencing Primer 1
24-mer  /  38% GC
1 binding site
6227 .. 6250  =  24 annealed bases
Tm  =  56°C
Cas9-Down
2774 .. 5389  =  2616 bp
872 amino acids  =  101.2 kDa
Product: downstream fragment of Streptococcus
pyogenes
Cas9
Cas9-Down
2774 .. 5389  =  2616 bp
872 amino acids  =  101.2 kDa
Product: downstream fragment of Streptococcus
pyogenes
Cas9
AmpR
660 .. 1520  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   660 .. 728  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
660 .. 1520  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   729 .. 1520  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
660 .. 1520  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1691 .. 2279  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1691 .. 2279  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hGH poly(A) signal
5461 .. 5937  =  477 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
5461 .. 5937  =  477 bp
human growth hormone polyadenylation signal
U6 promoter
6060 .. 6300  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6060 .. 6300  =  241 bp
RNA polymerase III promoter for human U6 snRNA
AAV2 ITR
153 .. 293  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
153 .. 293  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
2624 .. 2764  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
2624 .. 2764  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
555 .. 659  =  105 bp
AmpR promoter
555 .. 659  =  105 bp
gRNA scaffold
1 .. 86  =  86 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
1 .. 86  =  86 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
FLAG
5426 .. 5449  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
5426 .. 5449  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
5405 .. 5425  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5405 .. 5425  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
87 .. 92  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
87 .. 92  =  6 bp
RNA polymerase III transcription terminator
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