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pLenti4/BLOCK-iT™-DEST

Lentiviral Gateway® destination vector for an shRNA cassette from an H1/TO entry vector.

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pLenti4 BLOCK-iT-DEST Sequence and MappLenti4 BLOCK-iT-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PciI (7644) AhdI (6756) SfiI (5128) BbsI (4606) KpnI (4508) Acc65I (4504) SwaI (4499) PmlI (4485) FseI (4384) SgrAI (4222) AatII (4154) ZraI (4152) KasI (413) NarI (414) SfoI (415) PluTI (417) NruI * (609) EcoNI (942) MfeI (961) AleI (1349) KflI - PpuMI (1706) NheI (1797) BmtI (1801) AfeI (1802) SpeI (1827) BspEI (2303) BsmBI (2531) PasI - PflMI * (2539) BstZ17I (2842) SrfI (3178) BfuAI - BspMI (3422) SalI (3435) PaeR7I - PspXI - XhoI (3590) XbaI (3596) PspOMI (3602) ApaI (3606) SacII (3609) BstBI (3613) MluI (3657) AgeI (3663) MauBI (4144) pLenti4/BLOCK-iT™-DEST 8054 bp
PciI  (7644)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AhdI  (6756)
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.
SfiI  (5128)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
BbsI  (4606)
1 site
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.
KpnI  (4508)
1 site
G G T A C C C C A T G G
Acc65I  (4504)
1 site
G G T A C C C C A T G G
SwaI  (4499)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
PmlI  (4485)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FseI  (4384)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
SgrAI  (4222)
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.
AatII  (4154)
1 site
G A C G T C C T G C A G
ZraI  (4152)
1 site
G A C G T C C T G C A G
KasI  (413)
1 site
G G C G C C C C G C G G
NarI  (414)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (415)
1 site
G G C G C C C C G C G G
PluTI  (417)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
NruI  (609)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
EcoNI  (942)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MfeI  (961)
1 site
C A A T T G G T T A A C
AleI  (1349)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (1706)
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.
PpuMI  (1706)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
NheI  (1797)
1 site
G C T A G C C G A T C G
BmtI  (1801)
1 site
G C T A G C C G A T C G
AfeI  (1802)
1 site
A G C G C T T C G C G A
SpeI  (1827)
1 site
A C T A G T T G A T C A
BspEI  (2303)
1 site
T C C G G A A G G C C T
BsmBI  (2531)
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.
PasI  (2539)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PflMI  (2539)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BstZ17I  (2842)
1 site
G T A T A C C A T A T G
SrfI  (3178)
1 site
G C C C G G G C C G G G C C C G
BfuAI  (3422)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (3422)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
SalI  (3435)
1 site
G T C G A C C A G C T G
PaeR7I  (3590)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3590)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3590)
1 site
C T C G A G G A G C T C
XbaI  (3596)
1 site
T C T A G A A G A T C T
PspOMI  (3602)
1 site
G G G C C C C C C G G G
ApaI  (3606)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SacII  (3609)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BstBI  (3613)
1 site
T T C G A A A A G C T T
MluI  (3657)
1 site
A C G C G T T G C G C A
AgeI  (3663)
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.
MauBI  (4144)
1 site
C G C G C G C G G C G C G C G C
AmpR
5969 .. 6829  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5969 .. 6037  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5969 .. 6829  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6038 .. 6829  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5969 .. 6829  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
2094 .. 2753  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2094 .. 2753  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
7000 .. 7588  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7000 .. 7588  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5383 .. 5838  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5383 .. 5838  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BleoR
4108 .. 4482  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin
BleoR
4108 .. 4482  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin
SV40 promoter
3701 .. 4030  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
3701 .. 4030  =  330 bp
SV40 enhancer and early promoter
ccdB
3095 .. 3400  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
3095 .. 3400  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
RRE
1075 .. 1308  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1075 .. 1308  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
4575 .. 4808  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
4575 .. 4808  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RSV promoter
1 .. 229  =  229 bp
Rous Sarcoma Virus enhancer/promoter
RSV promoter
1 .. 229  =  229 bp
Rous Sarcoma Virus enhancer/promoter
5' LTR (truncated)
230 .. 410  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
230 .. 410  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 Ψ
457 .. 582  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
457 .. 582  =  126 bp
packaging signal of human immunodeficiency virus
type 1
attR1
1861 .. 1985  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1861 .. 1985  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3441 .. 3565  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3441 .. 3565  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
4880 .. 5001  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4880 .. 5001  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5864 .. 5968  =  105 bp
AmpR promoter
5864 .. 5968  =  105 bp
EM7 promoter
4042 .. 4089  =  48 bp
synthetic bacterial promoter
EM7 promoter
4042 .. 4089  =  48 bp
synthetic bacterial promoter
V5 tag
3618 .. 3659  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3618 .. 3659  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
2010 .. 2040  =  31 bp
   Segment 1:  -35  
   2010 .. 2015  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2010 .. 2040  =  31 bp
   Segment 2:  
   2016 .. 2033  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2010 .. 2040  =  31 bp
   Segment 3:  -10  
   2034 .. 2040  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2010 .. 2040  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
SV40 ori
3881 .. 4016  =  136 bp
SV40 origin of replication
SV40 ori
3881 .. 4016  =  136 bp
SV40 origin of replication
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