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Plasmid Files

lentiCRISPR v2

Zhang lab lentiviral transfer plasmid, also known as pLentiCRISPR v2, for CRISPR/Cas9 genome editing of mammalian cells with a single guide RNA (sgRNA).

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lentiCRISPR v2 Sequence and MaplentiCRISPR v2.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #52961
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 AmpR promoter FLAG hU6-F (2608 .. 2628) LKO.1 5' (2779 .. 2798) BsmBI (2852) BsmBI (4737) polIII terminator lentiCRISPR v2 14,873 bp
BsmBI  (2852)
2 sites
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI  (4737)
2 sites
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
hU6-F
21-mer  /  48% GC
1 binding site
2608 .. 2628  =  21 annealed bases
Tm  =  56°C
primer for sequencing an inserted sgRNA
LKO.1 5'
20-mer  /  40% GC
1 binding site
2779 .. 2798  =  20 annealed bases
Tm  =  51°C
primer for sequencing an inserted sgRNA
Cas9
5111 .. 9214  =  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
5111 .. 9214  =  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
filler
2853 .. 4737  =  1885 bp
digest with BsmBI to replace the filler with an
sgRNA  
filler
2853 .. 4737  =  1885 bp
digest with BsmBI to replace the filler with an
sgRNA  
AmpR
13,880 .. 14,740  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   13,880 .. 14,671  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
13,880 .. 14,740  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   14,672 .. 14,740  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
13,880 .. 14,740  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
9353 .. 9949  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
9353 .. 9949  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
9965 .. 10,553  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
9965 .. 10,553  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
13,121 .. 13,709  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
13,121 .. 13,709  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
11,160 .. 11,588  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
11,160 .. 11,588  =  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
12,045 .. 12,419  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
12,045 .. 12,419  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
SV40 promoter
11,602 .. 11,931  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
11,602 .. 11,931  =  330 bp
SV40 enhancer and early promoter
U6 promoter
2608 .. 2848  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
2608 .. 2848  =  241 bp
RNA polymerase III promoter for human U6 snRNA
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.
3' LTR (ΔU3)
10,625 .. 10,858  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
10,625 .. 10,858  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
bGH poly(A) signal
10,890 .. 11,114  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
10,890 .. 11,114  =  225 bp
bovine growth hormone polyadenylation signal
EF-1α core promoter
4875 .. 5086  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
4875 .. 5086  =  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
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
12,549 .. 12,670  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
12,549 .. 12,670  =  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
14,741 .. 14,845  =  105 bp
AmpR promoter
14,741 .. 14,845  =  105 bp
gRNA scaffold
4738 .. 4813  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
4738 .. 4813  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
P2A
9296 .. 9352  =  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
9296 .. 9352  =  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
9215 .. 9262  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
9215 .. 9262  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
EM7 promoter
11,979 .. 12,026  =  48 bp
synthetic bacterial promoter
EM7 promoter
11,979 .. 12,026  =  48 bp
synthetic bacterial promoter
FLAG
9263 .. 9286  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
9263 .. 9286  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
polIII terminator
4814 .. 4819  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
4814 .. 4819  =  6 bp
RNA polymerase III transcription terminator
SV40 ori
11,782 .. 11,917  =  136 bp
SV40 origin of replication
SV40 ori
11,782 .. 11,917  =  136 bp
SV40 origin of replication
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