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Plasmid Files

pCas-Guide-AAVS1

Mammalian guide RNA (gRNA) vector for transgene integration at the AAVS1 locus in the human genome. Use together with pAAVS1-puro-DNR.

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pCas-Guide-AAVS1.dna
Map and Sequence File:    Download    Open   
Sequence Author:  OriGene
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HpaI (8006) ScaI (7523) AlwNI (6562) HaeII (6394) BciVI (6349) PciI (6174) BstXI (5898) NsiI (5782) BbvCI (5747) SmaI (5463) TspMI - XmaI (5461) SacII (5435) FseI (5431) NaeI (5429) NgoMIV (5427) PmeI (5421) PaeR7I - PspXI - XhoI (5343) NotI (5337) BsiWI (5331) BstEII (5271) XcmI (4712) KflI (4550) BsmI (4145) DraIII - PmlI (40) BsrGI (445) SpeI (501) BtgZI (836) SnaBI (842) Eco53kI (1068) SacI (1070) SalI (1129) Acc65I (1142) KpnI (1146) BglII (1152) AsiSI (1168) BstZ17I (1387) BfuAI - BspMI (2241) PshAI (2748) SexAI * (3744) PflFI - Tth111I (3746) pCas-Guide-AAVS1 8009 bp
HpaI  (8006)
1 site
G T T A A C C A A T T G
ScaI  (7523)
1 site
A G T A C T T C A T G A
AlwNI  (6562)
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.
HaeII  (6394)
1 site
R G C G C Y Y C G C G R
BciVI  (6349)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
PciI  (6174)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstXI  (5898)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
NsiI  (5782)
1 site
A T G C A T T A C G T A
BbvCI  (5747)
1 site
C C T C A G C G G A G T C G
SmaI  (5463)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (5461)
1 site
C C C G G G G G G C C C
XmaI  (5461)
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.
SacII  (5435)
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.
FseI  (5431)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
NaeI  (5429)
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.
NgoMIV  (5427)
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.
PmeI  (5421)
1 site
G T T T A A A C C A A A T T T G
PaeR7I  (5343)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5343)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5343)
1 site
C T C G A G G A G C T C
NotI  (5337)
1 site
G C G G C C G C C G C C G G C G
BsiWI  (5331)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BstEII  (5271)
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.
XcmI  (4712)
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.
KflI  (4550)
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.
BsmI  (4145)
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.
DraIII  (40)
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.
PmlI  (40)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BsrGI  (445)
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.
SpeI  (501)
1 site
A C T A G T T G A T C A
BtgZI  (836)
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.
SnaBI  (842)
1 site
T A C G T A A T G C A T
Eco53kI  (1068)
1 site
G A G C T C C T C G A G
SacI  (1070)
1 site
G A G C T C C T C G A G
SalI  (1129)
1 site
G T C G A C C A G C T G
Acc65I  (1142)
1 site
G G T A C C C C A T G G
KpnI  (1146)
1 site
G G T A C C C C A T G G
BglII  (1152)
1 site
A G A T C T T C T A G A
AsiSI  (1168)
1 site
G C G A T C G C C G C T A G C G
BstZ17I  (1387)
1 site
G T A T A C C A T A T G
BfuAI  (2241)
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  (2241)
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.
PshAI  (2748)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
SexAI  (3744)
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.
PflFI  (3746)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (3746)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
Cas9
1173 .. 5276  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
1173 .. 5276  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
6970 .. 7830  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6970 .. 7761  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6970 .. 7830  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7762 .. 7830  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6970 .. 7830  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
hGH poly(A) signal
5464 .. 6086  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
5464 .. 6086  =  623 bp
human growth hormone polyadenylation signal
ori
6207 .. 6795  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6207 .. 6795  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
487 .. 866  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
487 .. 866  =  380 bp
human cytomegalovirus immediate early enhancer
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
867 .. 1070  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
867 .. 1070  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
gRNA scaffold
360 .. 435  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
360 .. 435  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
Myc
5346 .. 5375  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
5346 .. 5375  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
FLAG
5394 .. 5417  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
5394 .. 5417  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
SV40 NLS
5277 .. 5297  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5277 .. 5297  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5304 .. 5324  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5304 .. 5324  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
AAVS1 target sequence
340 .. 359  =  20 bp
AAVS1 target sequence
340 .. 359  =  20 bp
polIII terminator
436 .. 441  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
436 .. 441  =  6 bp
RNA polymerase III transcription terminator
ORF:  6176 .. 6532  =  357 bp
ORF:  118 amino acids  =  13.2 kDa
ORF:  7100 .. 7366  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1026 .. 5423  =  4398 bp
ORF:  1465 amino acids  =  169.8 kDa
ORF:  1431 .. 1673  =  243 bp
ORF:  80 amino acids  =  9.6 kDa
ORF:  3225 .. 3569  =  345 bp
ORF:  114 amino acids  =  13.5 kDa
ORF:  3723 .. 4127  =  405 bp
ORF:  134 amino acids  =  15.6 kDa
ORF:  4596 .. 5219  =  624 bp
ORF:  207 amino acids  =  24.3 kDa
ORF:  5828 .. 6052  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  6970 .. 7830  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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