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lenti MS2-P65-HSF1_Hygro

Zhang lab lentiviral vector for expressing a hygromycin resistance marker plus an MS2 RNA-binding domain fused to the p65 and HSF1 transcription factors.

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lenti MS2-P65-HSF1_Hygro Sequence and Maplenti MS2-P65-HSF1_Hygro.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #61426
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 SspI (11,620) ScaI (11,296) PvuI (11,186) FspI (11,038) AhdI (10,816) BleoR BmgBI (9042) SgrAI (9022) MauBI (8944) EM7 promoter PmeI (7731) 5' LTR (truncated) KpnI (7154) Acc65I (7150) Bsu36I (7146) BspDI - ClaI (7018) EcoRI (6404) RsrII (6261) SgrDI (1) MluI (231) SpeI (252) CMV enhancer NdeI (487) SnaBI (593) AleI (1954) KflI (2311) NheI (2601) BmtI (2605) AgeI (2765) BsiWI (3866) ATG BstXI (4063) BamHI (4352) BsrGI (5297) BsmBI (5437) lenti MS2-P65-HSF1_Hygro 11,736 bp
SspI  (11,620)
1 site
A A T A T T T T A T A A
ScaI  (11,296)
1 site
A G T A C T T C A T G A
PvuI  (11,186)
1 site
C G A T C G G C T A G C
FspI  (11,038)
1 site
T G C G C A A C G C G T
AhdI  (10,816)
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.
BmgBI  (9042)
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.
SgrAI  (9022)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
MauBI  (8944)
1 site
C G C G C G C G G C G C G C G C
PmeI  (7731)
1 site
G T T T A A A C C A A A T T T G
KpnI  (7154)
1 site
G G T A C C C C A T G G
Acc65I  (7150)
1 site
G G T A C C C C A T G G
Bsu36I  (7146)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BspDI  (7018)
1 site
A T C G A T T A G C T A
ClaI  (7018)
1 site
A T C G A T T A G C T A
EcoRI  (6404)
1 site
G A A T T C C T T A A G
RsrII  (6261)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
MluI  (231)
1 site
A C G C G T T G C G C A
SpeI  (252)
1 site
A C T A G T T G A T C A
NdeI  (487)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
SnaBI  (593)
1 site
T A C G T A A T G C A T
AleI  (1954)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (2311)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
NheI  (2601)
1 site
G C T A G C C G A T C G
BmtI  (2605)
1 site
G C T A G C C G A T C G
AgeI  (2765)
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.
BsiWI  (3866)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BstXI  (4063)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BamHI  (4352)
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.
BsrGI  (5297)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BsmBI  (5437)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
EF-1α promoter
2687 .. 3865  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
2687 .. 3865  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
HygR
5369 .. 6388  =  1020 bp
340 amino acids  =  37.9 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
5369 .. 6388  =  1020 bp
340 amino acids  =  37.9 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
10,743 .. 11,603  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,743 .. 11,534  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,743 .. 11,603  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   11,535 .. 11,603  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,743 .. 11,603  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
WPRE
6428 .. 7016  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
6428 .. 7016  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
9984 .. 10,572  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9984 .. 10,572  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
p65
4358 .. 4900  =  543 bp
181 amino acids  =  18.5 kDa
Product: C-terminal portion of the p65 subunit of
mouse NF-κB
p65
4358 .. 4900  =  543 bp
181 amino acids  =  18.5 kDa
Product: C-terminal portion of the p65 subunit of
mouse NF-κB
f1 ori
8023 .. 8451  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
8023 .. 8451  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
MS2-N55K
3881 .. 4267  =  387 bp
129 amino acids  =  13.7 kDa
Product: bacteriophage MS2 coat protein
binds with high affinity to a specific stem-loop
structure in the viral RNA (Lim et al., 1994)
MS2-N55K
3881 .. 4267  =  387 bp
129 amino acids  =  13.7 kDa
Product: bacteriophage MS2 coat protein
binds with high affinity to a specific stem-loop
structure in the viral RNA (Lim et al., 1994)
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
BleoR
8908 .. 9282  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
8908 .. 9282  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
HSF1
4925 .. 5296  =  372 bp
124 amino acids  =  13.0 kDa
Product: C-terminal activation domain from the
human heat shock transcription factor HSF1
HSF1
4925 .. 5296  =  372 bp
124 amino acids  =  13.0 kDa
Product: C-terminal activation domain from the
human heat shock transcription factor HSF1
SV40 promoter
8465 .. 8794  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
8465 .. 8794  =  330 bp
SV40 enhancer and early promoter
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1680 .. 1913  =  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
7753 .. 7977  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
7753 .. 7977  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
5' LTR (truncated)
835 .. 1015  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
835 .. 1015  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
7541 .. 7721  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
7541 .. 7721  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
9412 .. 9533  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
9412 .. 9533  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
11,604 .. 11,708  =  105 bp
AmpR promoter
11,604 .. 11,708  =  105 bp
T2A
5312 .. 5365  =  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
5312 .. 5365  =  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.
EM7 promoter
8842 .. 8889  =  48 bp
synthetic bacterial promoter
EM7 promoter
8842 .. 8889  =  48 bp
synthetic bacterial promoter
SV40 NLS
4322 .. 4342  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4322 .. 4342  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
3878 .. 3880  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3878 .. 3880  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
EF-1α intron A
2918 .. 3856  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
2918 .. 3856  =  939 bp
intron upstream of the start codon of human EF-1α
SV40 ori
8645 .. 8780  =  136 bp
SV40 origin of replication
SV40 ori
8645 .. 8780  =  136 bp
SV40 origin of replication
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