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Plasmid Files

pAAV-Guide-it-1 Vector (Linear)

Linearized CRISPR/Cas9 adeno-associated virus (AAV) vector with S. aureus Cas9, for cloning and expression of a single guide RNA (sgRNA).

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pAAV-Guide-it-1 Vector (Linear).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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End (7028) Guide-it™ Sequencing Primer 1 (6950 .. 6973) EcoRI (6777) SphI (6769) BspEI (6506) MfeI (6396) PsiI (6240) Bsu36I (5390) AvrII (5018) BstAPI (4924) BsmI (4891) AccI (4623) SalI (4622) BstEII (4282) SexAI * (4131) ApaI * (3983) PspOMI * (3979) StuI (3905) BlpI (3734) Start (0) EagI - NotI (83) BglII (90) KasI (401) NarI (402) SfoI (403) PluTI (405) XmnI (790) TsoI (992) DrdI (2176) PciI (2278) BspQI - SapI (2322) HindIII (2717) SnaBI (3055) AflII (3299) pAAV-Guide-it-1 Vector (Linear) 7028 bp
End  (7028)
0 sites
EcoRI  (6777)
1 site
G A A T T C C T T A A G
SphI  (6769)
1 site
G C A T G C C G T A C G
BspEI  (6506)
1 site
T C C G G A A G G C C T
MfeI  (6396)
1 site
C A A T T G G T T A A C
PsiI  (6240)
1 site
T T A T A A A A T A T T
Bsu36I  (5390)
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.
AvrII  (5018)
1 site
C C T A G G G G A T C C
BstAPI  (4924)
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.
BsmI  (4891)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
AccI  (4623)
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.
SalI  (4622)
1 site
G T C G A C C A G C T G
BstEII  (4282)
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.
SexAI  (4131)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
ApaI  (3983)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI can be used between 25°C and 37°C.
PspOMI  (3979)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
StuI  (3905)
1 site
A G G C C T T C C G G A
BlpI  (3734)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Start  (0)
0 sites
EagI  (83)
1 site
C G G C C G G C C G G C
NotI  (83)
1 site
G C G G C C G C C G C C G G C G
BglII  (90)
1 site
A G A T C T T C T A G A
KasI  (401)
1 site
G G C G C C C C G C G G
NarI  (402)
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  (403)
1 site
G G C G C C C C G C G G
PluTI  (405)
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.
XmnI  (790)
1 site
G A A N N N N T T C C T T N N N N A A G
TsoI  (992)
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  (2176)
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.
PciI  (2278)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2322)
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  (2322)
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.
HindIII  (2717)
1 site
A A G C T T T T C G A A
SnaBI  (3055)
1 site
T A C G T A A T G C A T
AflII  (3299)
1 site
C T T A A G G A A T T C
Guide-it™ Sequencing Primer 1
24-mer  /  38% GC
1 binding site
6950 .. 6973  =  24 annealed bases
Tm  =  56°C
SaCas9
3344 .. 6499  =  3156 bp
1052 amino acids  =  123.8 kDa
Product: Cas9 (Csn1) endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
SaCas9
3344 .. 6499  =  3156 bp
1052 amino acids  =  123.8 kDa
Product: Cas9 (Csn1) endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
603 .. 1463  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   603 .. 671  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
603 .. 1463  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   672 .. 1463  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
603 .. 1463  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1634 .. 2222  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1634 .. 2222  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
2776 .. 3079  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2776 .. 3079  =  304 bp
human cytomegalovirus immediate early enhancer
U6 promoter
6783 .. 7023  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6783 .. 7023  =  241 bp
RNA polymerase III promoter for human U6 snRNA
bGH poly(A) signal
6569 .. 6776  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
6569 .. 6776  =  208 bp
bovine growth hormone polyadenylation signal
CMV promoter
3080 .. 3283  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
3080 .. 3283  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
AAV2 ITR
96 .. 236  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AAV2 ITR
96 .. 236  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AAV2 ITR
2567 .. 2707  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AAV2 ITR
2567 .. 2707  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AmpR promoter
498 .. 602  =  105 bp
AmpR promoter
498 .. 602  =  105 bp
Sa gRNA scaffold
1 .. 76  =  76 bp
guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system
Sa gRNA scaffold
1 .. 76  =  76 bp
guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system
FLAG
6536 .. 6559  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
6536 .. 6559  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
SV40 NLS
3323 .. 3343  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
3323 .. 3343  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6515 .. 6535  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6515 .. 6535  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
Kozak sequence
3308 .. 3317  =  10 bp
vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)
Kozak sequence
3308 .. 3317  =  10 bp
vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)
polIII terminator
77 .. 81  =  5 bp
RNA polymerase III transcription terminator
polIII terminator
77 .. 81  =  5 bp
RNA polymerase III transcription terminator
ATG
3314 .. 3316  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3314 .. 3316  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  110 .. 391  =  282 bp
ORF:  93 amino acids  =  9.9 kDa
ORF:  3239 .. 6562  =  3324 bp
ORF:  1107 amino acids  =  130.0 kDa
ORF:  603 .. 1463  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3604 .. 3843  =  240 bp
ORF:  79 amino acids  =  8.5 kDa
ORF:  4705 .. 4962  =  258 bp
ORF:  85 amino acids  =  9.8 kDa
ORF:  6538 .. 6810  =  273 bp
ORF:  90 amino acids  =  10.0 kDa
ORF:  1067 .. 1333  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3287 .. 6478  =  3192 bp
ORF:  1063 amino acids  =  114.4 kDa
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