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Plasmid Files

pLV hUbC-Cas9-T2A-GFP

Lentiviral vector for co-expression of human codon-optimized Cas9 and enhanced GFP (EGFP).

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pLV hUbC-Cas9-T2A-GFP Sequence and MappLV hUbC-Cas9-T2A-GFP.dna
Map and Sequence File   
Sequence Author:  Gersbach Lab / Addgene #53190
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 AmpR promoter AhdI (13,852) BstZ17I (12,580) FseI (12,220) SgrAI (12,058) MauBI (11,980) EM7 promoter PmeI (10,767) AgeI (9391) BsrGI (9383) BmtI (8615) NheI (8611) SV40 NLS AscI (8429) FspAI (7424) SgrDI (1) MluI (231) SnaBI (593) NotI (1525) BbvCI (1801) AbsI - PspXI (2881) XbaI (4357) ATG SV40 NLS SbfI (4721) XcmI (4825) BlpI (5422) BamHI (6479) BstXI (7263) pLV hUbC-Cas9-T2A-GFP 14,772 bp
AhdI  (13,852)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BstZ17I  (12,580)
1 site
G T A T A C C A T A T G
FseI  (12,220)
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  (12,058)
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  (11,980)
1 site
C G C G C G C G G C G C G C G C
PmeI  (10,767)
1 site
G T T T A A A C C A A A T T T G
AgeI  (9391)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BsrGI  (9383)
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.
BmtI  (8615)
1 site
G C T A G C C G A T C G
NheI  (8611)
1 site
G C T A G C C G A T C G
AscI  (8429)
1 site
G G C G C G C C C C G C G C G G
FspAI  (7424)
1 site
R T G C G C A Y Y A C G C G T R
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
SnaBI  (593)
1 site
T A C G T A A T G C A T
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
AbsI  (2881)
1 site
C C T C G A G G G G A G C T C C
PspXI  (2881)
1 site
V C T C G A G B B G A G C T C V
XbaI  (4357)
1 site
T C T A G A A G A T C T
SbfI  (4721)
1 site
C C T G C A G G G G A C G T C C
XcmI  (4825)
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.
BlpI  (5422)
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.
BamHI  (6479)
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.
BstXI  (7263)
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.
Cas9
4474 .. 8577  =  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
4474 .. 8577  =  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
UbC promoter
3124 .. 4334  =  1211 bp
human ubiquitin C promoter
UbC promoter
3124 .. 4334  =  1211 bp
human ubiquitin C promoter
AmpR
13,779 .. 14,639  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   13,779 .. 14,570  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
13,779 .. 14,639  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   14,571 .. 14,639  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
13,779 .. 14,639  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
EGFP
8677 .. 9390  =  714 bp
238 amino acids  =  26.8 kDa
   Segment 1:  1a  
   8677 .. 8679  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
8677 .. 9390  =  714 bp
238 amino acids  =  26.8 kDa
   Segment 2:  
   8680 .. 9390  =  711 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
8677 .. 9390  =  714 bp
238 amino acids  =  26.8 kDa
2 segments
Product: enhanced GFP
mammalian codon-optimized
WPRE
9464 .. 10,052  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
9464 .. 10,052  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
13,020 .. 13,608  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
13,020 .. 13,608  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
11,059 .. 11,487  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
11,059 .. 11,487  =  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
11,944 .. 12,318  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
11,944 .. 12,318  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
SV40 promoter
11,501 .. 11,830  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
11,501 .. 11,830  =  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
10,789 .. 11,013  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
10,789 .. 11,013  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
LTR (truncated)
835 .. 1015  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
LTR (truncated)
835 .. 1015  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
LTR (truncated)
10,577 .. 10,757  =  181 bp
truncated long terminal repeat (LTR) from HIV-1
LTR (truncated)
10,577 .. 10,757  =  181 bp
truncated 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
12,448 .. 12,569  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
12,448 .. 12,569  =  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
14,640 .. 14,744  =  105 bp
AmpR promoter
14,640 .. 14,744  =  105 bp
3xFLAG
4372 .. 4437  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
4372 .. 4437  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
T2A
8617 .. 8670  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
8617 .. 8670  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
EM7 promoter
11,878 .. 11,925  =  48 bp
synthetic bacterial promoter
EM7 promoter
11,878 .. 11,925  =  48 bp
synthetic bacterial promoter
loxP
2602 .. 2635  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
loxP
2602 .. 2635  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
loxP
9406 .. 9439  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
loxP
9406 .. 9439  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
SV40 NLS
4444 .. 4464  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4444 .. 4464  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
8590 .. 8610  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
8590 .. 8610  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
4369 .. 4371  =  3 bp
1 amino acid  =  149.2 Da
ATG
4369 .. 4371  =  3 bp
1 amino acid  =  149.2 Da
SV40 ori
11,681 .. 11,816  =  136 bp
SV40 origin of replication
SV40 ori
11,681 .. 11,816  =  136 bp
SV40 origin of replication
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