pX330S-2

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

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.
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
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
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
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
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
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
polIII terminator
8611 .. 8616  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
8611 .. 8616  =  6 bp
RNA polymerase III transcription terminator
ORF:  757 .. 996  =  240 bp
ORF:  79 amino acids  =  9.1 kDa
ORF:  6409 .. 7419  =  1011 bp
ORF:  336 amino acids  =  37.5 kDa
ORF:  203 .. 550  =  348 bp
ORF:  115 amino acids  =  12.1 kDa
ORF:  5351 .. 5575  =  225 bp
ORF:  74 amino acids  =  8.3 kDa
ORF:  393 .. 704  =  312 bp
ORF:  103 amino acids  =  10.7 kDa
ORF:  819 .. 5090  =  4272 bp
ORF:  1423 amino acids  =  164.6 kDa
ORF:  2285 .. 2578  =  294 bp
ORF:  97 amino acids  =  10.8 kDa
ORF:  2849 .. 3298  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  7865 .. 8284  =  420 bp
ORF:  139 amino acids  =  15.5 kDa
ORF:  5251 .. 5532  =  282 bp
ORF:  93 amino acids  =  10.4 kDa
ORF:  6667 .. 7050  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
ORF:  7141 .. 7425  =  285 bp
ORF:  94 amino acids  =  10.5 kDa
ORF:  798 .. 3455  =  2658 bp
ORF:  885 amino acids  =  93.1 kDa
ORF:  6255 .. 6479  =  225 bp
ORF:  74 amino acids  =  8.6 kDa
ORF:  6660 .. 6917  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  6927 .. 7205  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
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