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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 Sequence and MappCas-Guide-AAVS1.dna
Map and Sequence File   
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) FLAG PaeR7I - PspXI - XhoI (5343) NotI (5337) BsiWI (5331) SV40 NLS SV40 NLS BstEII (5271) XcmI (4712) KflI (4550) BsmI (4145) DraIII - PmlI (40) AAVS1 target sequence polIII terminator BsrGI (445) SpeI (501) BtgZI (836) SnaBI (842) CMV promoter 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
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