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Plasmid Files

pX330A-1x2

Multiplex genome engineering plasmid for expressing a chimeric guide RNA (gRNA) together with Cas9, to be used for Golden Gate assembly.

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pX330A-1x2 Sequence and MappX330A-1x2.dna
Map and Sequence File   
Sequence Author:  Yamamoto Lab / Addgene #58766
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 lac operator BsaI (8500) polIII terminator BbsI - BpiI (8341) BbsI - BpiI (8319) nucleoplasmin NLS BsaI (8959) chicken β- actin promoter ATG pX330A-1x2 8964 bp
BsaI  (8500)
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.
BbsI  (8341)
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  (8341)
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  (8319)
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  (8319)
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  (8959)
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
AmpR
6394 .. 7254  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6394 .. 6462  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6394 .. 7254  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6463 .. 7254  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6394 .. 7254  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7425 .. 8013  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7425 .. 8013  =  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
lacZα
8503 .. 8808  =  306 bp
101 amino acids  =  11.2 kDa
Product: LacZα fragment of β-galactosidase
lacZα
8503 .. 8808  =  306 bp
101 amino acids  =  11.2 kDa
Product: LacZα fragment of β-galactosidase
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
8075 .. 8315  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
8075 .. 8315  =  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
AmpR promoter
6289 .. 6393  =  105 bp
AmpR promoter
6289 .. 6393  =  105 bp
gRNA scaffold
8342 .. 8417  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
8342 .. 8417  =  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
lac promoter
8852 .. 8882  =  31 bp
   Segment 3:  -10  
   8852 .. 8858  =  7 bp
promoter for the E. coli lac operon
lac promoter
8852 .. 8882  =  31 bp
   Segment 2:  
   8859 .. 8876  =  18 bp
promoter for the E. coli lac operon
lac promoter
8852 .. 8882  =  31 bp
   Segment 1:  -35  
   8877 .. 8882  =  6 bp
promoter for the E. coli lac operon
lac promoter
8852 .. 8882  =  31 bp
3 segments
promoter for the E. coli lac operon
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
lac operator
8828 .. 8844  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
8828 .. 8844  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
polIII terminator
8418 .. 8423  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
8418 .. 8423  =  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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