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Plasmid Files

pZIP-hCMV-RFP-Puro

Lentiviral vector with a human CMV promoter-driven cassette encoding TurboRFP plus a short hairpin RNA (shRNA) with an "UltramiR" microRNA scaffold.

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pZIP-hCMV-RFP-Puro Sequence and MappZIP-hCMV-RFP-Puro.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SgrDI (8375) SspI (8258) PvuI (7824) FspI (7676) AhdI (7454) DrdI (6672) PmeI (6196) PsiI (6146) PstI (5687) AscI (5069) MluI (5062) HpaI (4821) BamHI (4735) NotI (4725) BstEII (4258) RsrII (4240) SpeI (41) CMV enhancer NheI (405) BmtI (409) NruI * (867) BspQI - SapI (1136) FseI (1184) EcoNI (1204) MfeI (1223) BbvCI (1458) AleI (1611) KflI - PpuMI (1968) BspDI - ClaI (2214) AgeI (2825) BclI * (2847) BsrGI (2874) PasI (3055) SfiI (3300) PshAI (3464) EcoRI (3533) PspOMI (3659) ApaI (3663) PmlI (3862) AarI (3885) BmgBI (4089) PflFI - Tth111I (4166) BsiWI (4180) pZIP-hCMV-RFP-Puro 8377 bp
SgrDI  (8375)
1 site
C G T C G A C G G C A G C T G C
SspI  (8258)
1 site
A A T A T T T T A T A A
PvuI  (7824)
1 site
C G A T C G G C T A G C
FspI  (7676)
1 site
T G C G C A A C G C G T
AhdI  (7454)
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.
DrdI  (6672)
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.
PmeI  (6196)
1 site
G T T T A A A C C A A A T T T G
PsiI  (6146)
1 site
T T A T A A A A T A T T
PstI  (5687)
1 site
C T G C A G G A C G T C
AscI  (5069)
1 site
G G C G C G C C C C G C G C G G
MluI  (5062)
1 site
A C G C G T T G C G C A
HpaI  (4821)
1 site
G T T A A C C A A T T G
BamHI  (4735)
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.
NotI  (4725)
1 site
G C G G C C G C C G C C G G C G
BstEII  (4258)
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  (4240)
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.
SpeI  (41)
1 site
A C T A G T T G A T C A
NheI  (405)
1 site
G C T A G C C G A T C G
BmtI  (409)
1 site
G C T A G C C G A T C G
NruI  (867)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BspQI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
FseI  (1184)
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.
EcoNI  (1204)
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  (1223)
1 site
C A A T T G G T T A A C
BbvCI  (1458)
1 site
C C T C A G C G G A G T C G
AleI  (1611)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (1968)
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  (1968)
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.
BspDI  (2214)
1 site
A T C G A T T A G C T A
ClaI  (2214)
1 site
A T C G A T T A G C T A
AgeI  (2825)
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.
BclI  (2847)
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.
BsrGI  (2874)
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.
PasI  (3055)
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.
SfiI  (3300)
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.
PshAI  (3464)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
EcoRI  (3533)
1 site
G A A T T C C T T A A G
PspOMI  (3659)
1 site
G G G C C C C C C G G G
ApaI  (3663)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PmlI  (3862)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (3885)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BmgBI  (4089)
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.
PflFI  (4166)
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  (4166)
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.
BsiWI  (4180)
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.
AmpR
7381 .. 8241  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7381 .. 8172  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7381 .. 8241  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8173 .. 8241  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7381 .. 8241  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TurboRFP
2837 .. 3532  =  696 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea
quadricolor

mammalian codon-optimized
TurboRFP
2837 .. 3532  =  696 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea
quadricolor

mammalian codon-optimized
PuroR
4121 .. 4723  =  603 bp
200 amino acids  =  21.6 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
4121 .. 4723  =  603 bp
200 amino acids  =  21.6 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
5082 .. 5670  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
5082 .. 5670  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
6625 .. 7210  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6625 .. 7210  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3546 .. 4118  =  573 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3546 .. 4118  =  573 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
6207 .. 6536  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
6207 .. 6536  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
2235 .. 2538  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2235 .. 2538  =  304 bp
human cytomegalovirus immediate early enhancer
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
RRE
1337 .. 1570  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1337 .. 1570  =  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)
5878 .. 6111  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
5878 .. 6111  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
CMV promoter
2539 .. 2742  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2539 .. 2742  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
3' miR-30a
4935 .. 5061  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
3' miR-30a
4935 .. 5061  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
5' miR-30a
4744 .. 4865  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
4744 .. 4865  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
cPPT/CTS
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
8242 .. 8346  =  105 bp
AmpR promoter
8242 .. 8346  =  105 bp
shRNA
4866 .. 4934  =  69 bp
   Segment 1:  
   4866 .. 4892  =  27 bp
short hairpin RNA
shRNA
4866 .. 4934  =  69 bp
   Segment 2:  miR-30a loop  
   4893 .. 4907  =  15 bp
short hairpin RNA
shRNA
4866 .. 4934  =  69 bp
   Segment 3:  
   4908 .. 4934  =  27 bp
short hairpin RNA
shRNA
4866 .. 4934  =  69 bp
3 segments
short hairpin RNA
SV40 ori
6387 .. 6522  =  136 bp
SV40 origin of replication
SV40 ori
6387 .. 6522  =  136 bp
SV40 origin of replication
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