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Plasmid Files

lentiCas9-EGFP

Zhang lab lentiviral vector for expressing human codon-optimized S. pyogenes Cas9 plus enhanced GFP (EGFP).

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lentiCas9-EGFP Sequence and MaplentiCas9-EGFP.dna
Map and Sequence File   
Sequence Author:  Sharp & Zhang Labs / Addgene #63592
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 AmpR promoter SspI (13,061) PvuI (12,627) FspI (12,479) PciI (11,364) BstZ17I (10,985) FseI (10,625) SgrAI (10,463) MauBI (10,385) AvrII (10,220) StuI (10,219) PmeI (9172) KpnI (8595) Acc65I (8591) Bsu36I (8587) PaeR7I - XhoI (8473) BspDI - ClaI (8459) SacII (8372) EcoRI (7845) BsrGI (7834) BamHI (7062) FLAG SgrDI (1) MluI (231) SpeI (252) NdeI (487) SnaBI (593) KasI (1018) NarI (1019) SfoI (1020) PluTI (1022) NotI (1525) BbvCI (1801) NheI (2601) BmtI (2605) XbaI (2869) AfeI (2876) PasI (4655) BsaBI * (6384) lentiCas9-EGFP 13,177 bp
SspI  (13,061)
1 site
A A T A T T T T A T A A
PvuI  (12,627)
1 site
C G A T C G G C T A G C
FspI  (12,479)
1 site
T G C G C A A C G C G T
PciI  (11,364)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (10,985)
1 site
G T A T A C C A T A T G
FseI  (10,625)
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.
SgrAI  (10,463)
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.
MauBI  (10,385)
1 site
C G C G C G C G G C G C G C G C
AvrII  (10,220)
1 site
C C T A G G G G A T C C
StuI  (10,219)
1 site
A G G C C T T C C G G A
PmeI  (9172)
1 site
G T T T A A A C C A A A T T T G
KpnI  (8595)
1 site
G G T A C C C C A T G G
Acc65I  (8591)
1 site
G G T A C C C C A T G G
Bsu36I  (8587)
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.
PaeR7I  (8473)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (8473)
1 site
C T C G A G G A G C T C
BspDI  (8459)
1 site
A T C G A T T A G C T A
ClaI  (8459)
1 site
A T C G A T T A G C T A
SacII  (8372)
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.
EcoRI  (7845)
1 site
G A A T T C C T T A A G
BsrGI  (7834)
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.
BamHI  (7062)
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.
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
MluI  (231)
1 site
A C G C G T T G C G C A
SpeI  (252)
1 site
A C T A G T T G A T C A
NdeI  (487)
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.
SnaBI  (593)
1 site
T A C G T A A T G C A T
KasI  (1018)
1 site
G G C G C C C C G C G G
NarI  (1019)
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  (1020)
1 site
G G C G C C C C G C G G
PluTI  (1022)
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.
NotI  (1525)
1 site
G C G G C C G C C G C C G G C G
BbvCI  (1801)
1 site
C C T C A G C G G A G T C G
NheI  (2601)
1 site
G C T A G C C G A T C G
BmtI  (2605)
1 site
G C T A G C C G A T C G
XbaI  (2869)
1 site
T C T A G A A G A T C T
AfeI  (2876)
1 site
A G C G C T T C G C G A
PasI  (4655)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
BsaBI  (6384)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
Cas9
2886 .. 6989  =  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
2886 .. 6989  =  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
12,184 .. 13,044  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   12,184 .. 12,975  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
12,184 .. 13,044  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   12,976 .. 13,044  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
12,184 .. 13,044  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
EGFP
7128 .. 7844  =  717 bp
238 amino acids  =  26.8 kDa
   Segment 1:  1a  
   7128 .. 7130  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
7128 .. 7844  =  717 bp
238 amino acids  =  26.8 kDa
   Segment 2:  
   7131 .. 7844  =  714 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
7128 .. 7844  =  717 bp
238 amino acids  =  26.8 kDa
2 segments
Product: enhanced GFP
mammalian codon-optimized
WPRE
7869 .. 8457  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7869 .. 8457  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
11,425 .. 12,013  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
11,425 .. 12,013  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
9464 .. 9892  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
9464 .. 9892  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
BleoR
10,349 .. 10,723  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
10,349 .. 10,723  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
SV40 promoter
9906 .. 10,235  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
9906 .. 10,235  =  330 bp
SV40 enhancer and early promoter
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
bGH poly(A) signal
9194 .. 9418  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
9194 .. 9418  =  225 bp
bovine growth hormone polyadenylation signal
EF-1α core promoter
2650 .. 2861  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
2650 .. 2861  =  212 bp
core promoter for human elongation factor EF-1α
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
5' LTR (truncated)
835 .. 1015  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
835 .. 1015  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
8982 .. 9162  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
8982 .. 9162  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
10,853 .. 10,974  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
10,853 .. 10,974  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
13,045 .. 13,149  =  105 bp
AmpR promoter
13,045 .. 13,149  =  105 bp
P2A
7071 .. 7127  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
P2A
7071 .. 7127  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
nucleoplasmin NLS
6990 .. 7037  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6990 .. 7037  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
EM7 promoter
10,283 .. 10,330  =  48 bp
synthetic bacterial promoter
EM7 promoter
10,283 .. 10,330  =  48 bp
synthetic bacterial promoter
FLAG
7038 .. 7061  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
7038 .. 7061  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 ori
10,086 .. 10,221  =  136 bp
SV40 origin of replication
SV40 ori
10,086 .. 10,221  =  136 bp
SV40 origin of replication
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