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Plasmid Files

pKLV-U6gRNA(BbsI)-PGKpuro2ABFP

Vector for expressing a single guide RNA (sgRNA) together with TagBFP and a puromycin resistance marker.

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pKLV-U6gRNA(BbsI)-PGKpuro2ABFP Sequence and MappKLV-U6gRNA(BbsI)-PGKpuro2ABFP.dna
Map and Sequence File   
Sequence Author:  Yusa Lab / Addgene #50946
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 CMV enhancer BbsI (3291) BbsI (3313) gRNA scaffold polIII terminator pKLV-U6gRNA(BbsI)-PGKpuro2ABFP 8118 bp
BbsI  (3291)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (3313)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
AmpR
7130 .. 7990  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7130 .. 7921  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7130 .. 7990  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7922 .. 7990  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7130 .. 7990  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagBFP
4618 .. 5319  =  702 bp
233 amino acids  =  26.3 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
TagBFP
4618 .. 5319  =  702 bp
233 amino acids  =  26.3 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
PuroR
3949 .. 4545  =  597 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
3949 .. 4545  =  597 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
6371 .. 6959  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6371 .. 6959  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
PGK promoter
3410 .. 3909  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
3410 .. 3909  =  500 bp
mouse phosphoglycerate kinase 1 promoter
f1 ori
3 .. 458  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3 .. 458  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
715 .. 1018  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
715 .. 1018  =  304 bp
human cytomegalovirus immediate early enhancer
U6 promoter
3047 .. 3287  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
3047 .. 3287  =  241 bp
RNA polymerase III promoter for human U6 snRNA
RRE
2087 .. 2320  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
2087 .. 2320  =  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
5680 .. 5904  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5680 .. 5904  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
1020 .. 1218  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
1020 .. 1218  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
5' LTR (truncated)
1236 .. 1416  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
1236 .. 1416  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 Ψ
1463 .. 1588  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
1463 .. 1588  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
2847 .. 2964  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2847 .. 2964  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
7991 .. 8095  =  105 bp
AmpR promoter
7991 .. 8095  =  105 bp
gRNA scaffold
3312 .. 3387  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
3312 .. 3387  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
T2A
4564 .. 4617  =  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
4564 .. 4617  =  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.
polIII terminator
3388 .. 3393  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
3388 .. 3393  =  6 bp
RNA polymerase III transcription terminator
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