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Plasmid Files

pCas

Bacterial plasmid with a temperature-sensitive replicon encoding Cas9, an inducible single guide RNA (sgRNA), and the λ Red recombination system.

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pCas Sequence and MappCas.dna
Map and Sequence File   
Sequence Author:  Yang Lab / Addgene #62225
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 BglII (0) SgrAI (11,788) BglI (11,291) BsrGI (11,117) SalI (10,868) BspQI - SapI (10,120) AarI (9147) XbaI (9054) PmlI (7209) KpnI (6694) Acc65I (6690) lac operator PspOMI (924) ApaI (928) SnaBI (1879) SpeI (2715) EagI - NotI (3035) AsiSI - PvuI (3957) EcoNI (4042) PaeR7I - PspXI - XhoI (4353) SbfI (4504) BstZ17I (5131) BmgBI (5136) SwaI (5437) BlpI (5596) NheI (6015) BmtI (6019) BstBI (6254) pCas 12,545 bp
BglII  (0)
1 site
A G A T C T T C T A G A
SgrAI  (11,788)
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.
BglI  (11,291)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
BsrGI  (11,117)
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.
SalI  (10,868)
1 site
G T C G A C C A G C T G
BspQI  (10,120)
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  (10,120)
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.
AarI  (9147)
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.
XbaI  (9054)
1 site
T C T A G A A G A T C T
PmlI  (7209)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
KpnI  (6694)
1 site
G G T A C C C C A T G G
Acc65I  (6690)
1 site
G G T A C C C C A T G G
PspOMI  (924)
1 site
G G G C C C C C C G G G
ApaI  (928)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SnaBI  (1879)
1 site
T A C G T A A T G C A T
SpeI  (2715)
1 site
A C T A G T T G A T C A
EagI  (3035)
1 site
C G G C C G G C C G G C
NotI  (3035)
1 site
G C G G C C G C C G C C G G C G
AsiSI  (3957)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3957)
1 site
C G A T C G G C T A G C
EcoNI  (4042)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PaeR7I  (4353)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4353)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4353)
1 site
C T C G A G G A G C T C
SbfI  (4504)
1 site
C C T G C A G G G G A C G T C C
BstZ17I  (5131)
1 site
G T A T A C C A T A T G
BmgBI  (5136)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
SwaI  (5437)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BlpI  (5596)
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.
NheI  (6015)
1 site
G C T A G C C G A T C G
BmtI  (6019)
1 site
G C T A G C C G A T C G
BstBI  (6254)
1 site
T T C G A A A A G C T T
Cas9
4917 .. 9023  =  4107 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
4917 .. 9023  =  4107 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
lacI
404 .. 1486  =  1083 bp
360 amino acids  =  38.5 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
404 .. 1486  =  1083 bp
360 amino acids  =  38.5 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
Rep101(Ts)
1698 .. 2648  =  951 bp
316 amino acids  =  37.4 kDa
Product: temperature-sensitive version of the RepA
protein needed for replication with the pSC101
origin (Armstrong et al., 1984)
Rep101(Ts)
1698 .. 2648  =  951 bp
316 amino acids  =  37.4 kDa
Product: temperature-sensitive version of the RepA
protein needed for replication with the pSC101
origin (Armstrong et al., 1984)
araC
9153 .. 10,031  =  879 bp
292 amino acids  =  33.4 kDa
Product: L-arabinose regulatory protein
araC
9153 .. 10,031  =  879 bp
292 amino acids  =  33.4 kDa
Product: L-arabinose regulatory protein
KanR
3572 .. 4387  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
3572 .. 4387  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
Beta
10,808 .. 11,593  =  786 bp
261 amino acids  =  29.7 kDa
Product: single-stranded DNA binding recombinase
in the λ Red system
mediates pairing of a single-stranded DNA with a
complementary target
Beta
10,808 .. 11,593  =  786 bp
261 amino acids  =  29.7 kDa
Product: single-stranded DNA binding recombinase
in the λ Red system
mediates pairing of a single-stranded DNA with a
complementary target
Gam
10,386 .. 10,802  =  417 bp
138 amino acids  =  16.3 kDa
Product: inhibitor of the host RecBCD nuclease in
the λ Red system
protects the double-stranded DNA substrate for
recombination
Gam
10,386 .. 10,802  =  417 bp
138 amino acids  =  16.3 kDa
Product: inhibitor of the host RecBCD nuclease in
the λ Red system
protects the double-stranded DNA substrate for
recombination
araBAD promoter
10,058 .. 10,342  =  285 bp
promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)
araBAD promoter
10,058 .. 10,342  =  285 bp
promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)
λ tL3 terminator
12,271 .. 12,515  =  245 bp
transcription terminator tL3 from phage λ
λ tL3 terminator
12,271 .. 12,515  =  245 bp
transcription terminator tL3 from phage λ
pSC101 ori
2696 .. 2918  =  223 bp
low-copy replication origin that requires the Rep101
protein
pSC101 ori
2696 .. 2918  =  223 bp
low-copy replication origin that requires the Rep101
protein
tracrRNA
4543 .. 4621  =  79 bp
trans-activating CRISPR RNA for the Streptococcus
pyogenes
CRISPR/Cas9 system
tracrRNA
4543 .. 4621  =  79 bp
trans-activating CRISPR RNA for the Streptococcus
pyogenes
CRISPR/Cas9 system
lacIq promoter
1487 .. 1564  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
lacIq promoter
1487 .. 1564  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
gRNA scaffold
14 .. 89  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
14 .. 89  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
trc promoter
143 .. 172  =  30 bp
   Segment 3:  -10  
   143 .. 149  =  7 bp
strong E. coli promoter; hybrid between the trp and
lac UV5 promoters
trc promoter
143 .. 172  =  30 bp
   Segment 2:  
   150 .. 166  =  17 bp
strong E. coli promoter; hybrid between the trp and
lac UV5 promoters
trc promoter
143 .. 172  =  30 bp
   Segment 1:  -35  
   167 .. 172  =  6 bp
strong E. coli promoter; hybrid between the trp and
lac UV5 promoters
trc promoter
143 .. 172  =  30 bp
3 segments
strong E. coli promoter; hybrid between the trp and
lac UV5 promoters
lac operator
119 .. 135  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
119 .. 135  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
Exo
11,590 .. 12,270  =  681 bp
226 amino acids  =  25.9 kDa
Product: 5' to 3' double-stranded DNA exonuclease
in the λ Red system
creates 3' single-stranded DNA tails
Exo
11,590 .. 12,270  =  681 bp
226 amino acids  =  25.9 kDa
Product: 5' to 3' double-stranded DNA exonuclease
in the λ Red system
creates 3' single-stranded DNA tails
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