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GeneArt® CRISPR Nuclease Vector - CD4 (linearized)

All-in-one CRISPR/Cas9 vector for genome editing, with a CD4 reporter for bead-based cell isolation.

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GeneArt CRISPR Nuclease Vector - CD4 (linearized) Sequence and MapGeneArt CRISPR Nuclease Vector - CD4 (linearized).dna
Map and Sequence File   
Sequence Author:  Thermo Fisher Scientific (Invitrogen)
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 SgrDI (2281) AmpR promoter PvuI (1730) AhdI (1360) BstZ17I (88) Start (5) End (9803) PacI (9537) PsiI (9464) PmeI (8794) AgeI (8776) BmtI (8110) NheI (8106) AflII (7798) MluI (2511) SpeI (2532) SnaBI (2873) NotI (3191) ATG SV40 NLS Acc65I (3369) KpnI (3373) PflFI - Tth111I (3664) BamHI (4631) PspOMI (4647) ApaI (4651) BssHII (4751) AleI (4867) PaeR7I - PspXI - XhoI (5137) BmgBI (5802) PmlI (6081) XcmI (6831) AfeI (6939) BbvCI (7162) SV40 NLS EcoRI (7429) XbaI (7435) GeneArt® CRISPR Nuclease Vector - CD4 9798 bp
SgrDI  (2281)
1 site
C G T C G A C G G C A G C T G C
PvuI  (1730)
1 site
C G A T C G G C T A G C
AhdI  (1360)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BstZ17I  (88)
1 site
G T A T A C C A T A T G
Start  (5)
0 sites
End  (9803)
0 sites
PacI  (9537)
1 site
T T A A T T A A A A T T A A T T
PsiI  (9464)
1 site
T T A T A A A A T A T T
PmeI  (8794)
1 site
G T T T A A A C C A A A T T T G
AgeI  (8776)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BmtI  (8110)
1 site
G C T A G C C G A T C G
NheI  (8106)
1 site
G C T A G C C G A T C G
AflII  (7798)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
MluI  (2511)
1 site
A C G C G T T G C G C A
SpeI  (2532)
1 site
A C T A G T T G A T C A
SnaBI  (2873)
1 site
T A C G T A A T G C A T
NotI  (3191)
1 site
G C G G C C G C C G C C G G C G
Acc65I  (3369)
1 site
G G T A C C C C A T G G
KpnI  (3373)
1 site
G G T A C C C C A T G G
PflFI  (3664)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (3664)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BamHI  (4631)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
PspOMI  (4647)
1 site
G G G C C C C C C G G G
ApaI  (4651)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (4751)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
AleI  (4867)
1 site
C A C N N N N G T G G T G N N N N C A C
PaeR7I  (5137)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5137)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5137)
1 site
C T C G A G G A G C T C
BmgBI  (5802)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
PmlI  (6081)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
XcmI  (6831)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
AfeI  (6939)
1 site
A G C G C T T C G C G A
BbvCI  (7162)
1 site
C C T C A G C G G A G T C G
EcoRI  (7429)
1 site
G A A T T C C T T A A G
XbaI  (7435)
1 site
T C T A G A A G A T C T
Cas9
3295 .. 7395  =  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
3295 .. 7395  =  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
CD4 (truncated)
7504 .. 8775  =  1272 bp
424 amino acids  =  47.0 kDa
   Segment 1:  signal peptide  
   7504 .. 7578  =  75 bp
   25 amino acids  =  2.7 kDa
Product: truncated human CD4 cell surface
glycoprotein lacking the cytoplasmic tail
CD4 (truncated)
7504 .. 8775  =  1272 bp
424 amino acids  =  47.0 kDa
   Segment 2:  
   7579 .. 8775  =  1197 bp
   399 amino acids  =  44.3 kDa
Product: truncated human CD4 cell surface
glycoprotein lacking the cytoplasmic tail
CD4 (truncated)
7504 .. 8775  =  1272 bp
424 amino acids  =  47.0 kDa
2 segments
Product: truncated human CD4 cell surface
glycoprotein lacking the cytoplasmic tail
AmpR
1287 .. 2147  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1287 .. 2078  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1287 .. 2147  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2079 .. 2147  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1287 .. 2147  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
528 .. 1116  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
528 .. 1116  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
9106 .. 9534  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
9106 .. 9534  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
2518 .. 2897  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2518 .. 2897  =  380 bp
human cytomegalovirus immediate early enhancer
U6 promoter
9554 .. 9794  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
9554 .. 9794  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
2898 .. 3101  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2898 .. 3101  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
AmpR promoter
2148 .. 2252  =  105 bp
AmpR promoter
2148 .. 2252  =  105 bp
gRNA scaffold
1 .. 76  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
1 .. 76  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
T2A
7450 .. 7503  =  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
7450 .. 7503  =  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.
HSV TK poly(A) signal
8856 .. 8904  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
8856 .. 8904  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
V5 tag
3205 .. 3246  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3205 .. 3246  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
SV40 NLS
3250 .. 3270  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
3250 .. 3270  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7408 .. 7428  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7408 .. 7428  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
77 .. 82  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
77 .. 82  =  6 bp
RNA polymerase III transcription terminator
ATG
3202 .. 3204  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3202 .. 3204  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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