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Plasmid Files

pZIP-TRE3G-ZsGreen-Puro

Lentiviral vector with a doxycycline-inducible cassette encoding ZsGreen plus a short hairpin RNA (shRNA) with an "UltramiR" microRNA scaffold.

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pZIP-TRE3G-ZsGreen-Puro Sequence and MappZIP-TRE3G-ZsGreen-Puro.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SgrDI (9953) SspI (9836) DrdI (8250) PciI (8142) BmtI (7758) NheI (7754) KpnI (6972) Acc65I (6968) Bsu36I (6964) PstI (6848) PflMI (6484) MauBI (6228) BstEII (5760) RsrII (5742) BsiWI (5682) AvrII (5602) SfiI (5555) FseI (1150) BbvCI - Bpu10I (1424) AleI (1577) KflI - PpuMI (1934) cPPT/CTS MluI (2472) shRNA HpaI (2726) 5' miR-30a I-SceI (2820) NotI (2867) BsrGI (3019) BclI * (3487) BamHI (3578) AgeI (3590) BstZ17I (3598) BstXI (4005) BlpI (4338) AscI (4347) pZIP-TRE3G-ZsGreen-Puro 10,321 bp
SgrDI  (9953)
1 site
C G T C G A C G G C A G C T G C
SspI  (9836)
1 site
A A T A T T T T A T A A
DrdI  (8250)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PciI  (8142)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BmtI  (7758)
1 site
G C T A G C C G A T C G
NheI  (7754)
1 site
G C T A G C C G A T C G
KpnI  (6972)
1 site
G G T A C C C C A T G G
Acc65I  (6968)
1 site
G G T A C C C C A T G G
Bsu36I  (6964)
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.
PstI  (6848)
1 site
C T G C A G G A C G T C
PflMI  (6484)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
MauBI  (6228)
1 site
C G C G C G C G G C G C G C G C
BstEII  (5760)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
RsrII  (5742)
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.
BsiWI  (5682)
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.
AvrII  (5602)
1 site
C C T A G G G G A T C C
SfiI  (5555)
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.
FseI  (1150)
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.
BbvCI  (1424)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1424)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
AleI  (1577)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (1934)
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  (1934)
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.
MluI  (2472)
1 site
A C G C G T T G C G C A
HpaI  (2726)
1 site
G T T A A C C A A T T G
I-SceI  (2820)
1 site
T A G G G A T A A C A G G G T A A T A T C C C T A T T G T C C C A T T A

I-SceI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
After cleavage, I-SceI can remain bound to DNA and alter its
electrophoretic mobility.
NotI  (2867)
1 site
G C G G C C G C C G C C G G C G
BsrGI  (3019)
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.
BclI  (3487)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BamHI  (3578)
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.
AgeI  (3590)
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.
BstZ17I  (3598)
1 site
G T A T A C C A T A T G
BstXI  (4005)
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.
BlpI  (4338)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
AscI  (4347)
1 site
G G C G C G C C C C G C G C G G
AmpR
8959 .. 9819  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8959 .. 9750  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8959 .. 9819  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9751 .. 9819  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8959 .. 9819  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
Tet-On® 3G
4531 .. 5277  =  747 bp
248 amino acids  =  27.7 kDa
Product: modified rtTA protein that binds tightly to
promoters containing the tet operator in the
presence of doxycycline
Tet-On® 3G
4531 .. 5277  =  747 bp
248 amino acids  =  27.7 kDa
Product: modified rtTA protein that binds tightly to
promoters containing the tet operator in the
presence of doxycycline
ZsGreen1
2879 .. 3574  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
2879 .. 3574  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
LTR
1 .. 634  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
1 .. 634  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
7039 .. 7672  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
7039 .. 7672  =  634 bp
long terminal repeat (LTR) from HIV-1
PuroR
5626 .. 6225  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
5626 .. 6225  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
6243 .. 6831  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
6243 .. 6831  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
8203 .. 8788  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8203 .. 8788  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hPGK promoter
4002 .. 4512  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
4002 .. 4512  =  511 bp
human phosphoglycerate kinase 1 promoter
TRE3GS promoter
3614 .. 3981  =  368 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On® 3G, modified to
eliminate binding sites for endogenous mammalian
transcription factors
TRE3GS promoter
3614 .. 3981  =  368 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On® 3G, modified to
eliminate binding sites for endogenous mammalian
transcription factors
SV40 promoter
5288 .. 5617  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5288 .. 5617  =  330 bp
SV40 enhancer and early promoter
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
SV40 poly(A) signal
2187 .. 2321  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2187 .. 2321  =  135 bp
SV40 polyadenylation signal
3' miR-30a
2487 .. 2613  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
3' miR-30a
2487 .. 2613  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
5' miR-30a
2683 .. 2804  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
2683 .. 2804  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
cPPT/CTS
2028 .. 2143  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2028 .. 2143  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
9820 .. 9924  =  105 bp
AmpR promoter
9820 .. 9924  =  105 bp
shRNA
2614 .. 2682  =  69 bp
   Segment 3:  
   2614 .. 2640  =  27 bp
short hairpin RNA
shRNA
2614 .. 2682  =  69 bp
   Segment 2:  miR-30a loop  
   2641 .. 2655  =  15 bp
short hairpin RNA
shRNA
2614 .. 2682  =  69 bp
   Segment 1:  
   2656 .. 2682  =  27 bp
short hairpin RNA
shRNA
2614 .. 2682  =  69 bp
3 segments
short hairpin RNA
SV40 ori
5468 .. 5603  =  136 bp
SV40 origin of replication
SV40 ori
5468 .. 5603  =  136 bp
SV40 origin of replication
tet operator
3739 .. 3757  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3739 .. 3757  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3775 .. 3793  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3775 .. 3793  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3811 .. 3829  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3811 .. 3829  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3847 .. 3865  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3847 .. 3865  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3883 .. 3901  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3883 .. 3901  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3919 .. 3937  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3919 .. 3937  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3955 .. 3973  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3955 .. 3973  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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