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Plasmid Files

pX330S-2

Multiplex genome engineering plasmid for generating a chimeric guide RNA (gRNA) insert to be used for Golden Gate assembly.

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pX330S-2 Sequence and MappX330S-2.dna
Map and Sequence File   
Sequence Author:  Yamamoto Lab / Addgene #58778
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 polIII terminator BbsI - BpiI (8534) BbsI - BpiI (8512) BsaI (8253) nucleoplasmin NLS BsaI (8694) chicken β- actin promoter ATG pX330S-2 8698 bp
BbsI  (8534)
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.
BpiI  (8534)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
BbsI  (8512)
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.
BpiI  (8512)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
BsaI  (8253)
2 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BsaI  (8694)
2 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
Cas9
939 .. 5039  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
939 .. 5039  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
SmR
6628 .. 7419  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
6628 .. 7419  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
ori
7602 .. 8190  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7602 .. 8190  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5552 .. 6007  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5552 .. 6007  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
8 .. 293  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
8 .. 293  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
295 .. 572  =  278 bp
chicken β-actin promoter
295 .. 572  =  278 bp
U6 promoter
8268 .. 8508  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
8268 .. 8508  =  241 bp
RNA polymerase III promoter for human U6 snRNA
hybrid intron
573 .. 801  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
573 .. 801  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
5121 .. 5328  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5121 .. 5328  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
5337 .. 5477  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
5337 .. 5477  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
gRNA scaffold
8535 .. 8610  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
8535 .. 8610  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
3xFLAG
822 .. 887  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
822 .. 887  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
nucleoplasmin NLS
5040 .. 5087  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
5040 .. 5087  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
894 .. 914  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
894 .. 914  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
8611 .. 8616  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
8611 .. 8616  =  6 bp
RNA polymerase III transcription terminator
ATG
819 .. 821  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
819 .. 821  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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