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Plasmid Files

pLKO.1-puro U6 sgRNA BfuAI stuffer

Lentiviral vector for expressing a single guide RNA (sgRNA) by inserting a target sequence between BfuAI sites.

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pLKO.1-puro U6 sgRNA BfuAI stuffer Sequence and MappLKO.1-puro U6 sgRNA BfuAI stuffer.dna
Map and Sequence File   
Sequence Author:  Maehr & Wolfe Labs / Addgene #50920
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 BfuAI (6851) BfuAI (6815) U6 promoter polIII terminator 3' LTR ( Δ U3) pLKO.1-puro U6 sgRNA BfuAI stuffer 7145 bp
BfuAI  (6851)
2 sites
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.
BfuAI  (6815)
2 sites
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.
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2650 .. 2718  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2719 .. 3510  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2650 .. 3510  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
529 .. 1128  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
529 .. 1128  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
3681 .. 4269  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3681 .. 4269  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hPGK promoter
7142 .. 507  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
7142 .. 507  =  511 bp
human phosphoglycerate kinase 1 promoter
f1 ori
2064 .. 2519  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2064 .. 2519  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
U6 promoter
6570 .. 6810  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6570 .. 6810  =  241 bp
RNA polymerase III promoter for human U6 snRNA
3' LTR (ΔU3)
1256 .. 1489  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
1256 .. 1489  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RRE
5810 .. 6043  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
5810 .. 6043  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RSV promoter
4738 .. 4964  =  227 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
4738 .. 4964  =  227 bp
Rous sarcoma virus enhancer/promoter
5' LTR (truncated)
4965 .. 5145  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
4965 .. 5145  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
1722 .. 1857  =  136 bp
SV40 origin of replication
SV40 ori
1722 .. 1857  =  136 bp
SV40 origin of replication
HIV-1 Ψ
5192 .. 5317  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
5192 .. 5317  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
1561 .. 1682  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1561 .. 1682  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
6976 .. 7093  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
6976 .. 7093  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
2545 .. 2649  =  105 bp
AmpR promoter
2545 .. 2649  =  105 bp
gRNA scaffold
6852 .. 6927  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
6852 .. 6927  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
polIII terminator
6928 .. 6933  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
6928 .. 6933  =  6 bp
RNA polymerase III transcription terminator
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