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Plasmid Files

pSIREN-RetroQ-ZsGreen1

Linearized retroviral vector for inserting an shRNA cassette that will be co-expressed with ZsGreen1.

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pSIREN-RetroQ-ZsGreen1 Sequence and MappSIREN-RetroQ-ZsGreen1.dna
Map and Sequence File   
Sequence Author:  Clontech
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 < BamHI > (6562) U6 forward sequencing primer (6486 .. 6509) BglII (6303) Bpu10I (6182) BstEII (5979) ApoI (5764) EagI (5616) PshAI (5403) AscI (5269) CMV enhancer SspI (4382) XmnI (4177) ScaI (4058) PvuI (3948) FspI (3800) < EcoRI > (0) AgeI (605) BclI * (700) SgrAI (705) NgoMIV (882) NaeI (884) BmgBI (1109) BfuAI - BspMI (1129) PaeR7I - XhoI (1314) EcoRV (1321) PvuII (1364) SfiI (2391) StuI (2437) AvrII (2438) HindIII (2454) BspQI - SapI (2569) AflIII - PciI (2685) AlwNI (3101) pSIREN-RetroQ-ZsGreen1 6562 bp
End  (6562)
0 sites
BglII  (6303)
1 site
A G A T C T T C T A G A
Bpu10I  (6182)
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.
BstEII  (5979)
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.
ApoI  (5764)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EagI  (5616)
1 site
C G G C C G G C C G G C
PshAI  (5403)
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.
AscI  (5269)
1 site
G G C G C G C C C C G C G C G G
SspI  (4382)
1 site
A A T A T T T T A T A A
XmnI  (4177)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (4058)
1 site
A G T A C T T C A T G A
PvuI  (3948)
1 site
C G A T C G G C T A G C
FspI  (3800)
1 site
T G C G C A A C G C G T
Start  (0)
0 sites
AgeI  (605)
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  (700)
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.
SgrAI  (705)
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.
NgoMIV  (882)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (884)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
BmgBI  (1109)
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.
BfuAI  (1129)
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  (1129)
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.
PaeR7I  (1314)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1314)
1 site
C T C G A G G A G C T C
EcoRV  (1321)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
PvuII  (1364)
1 site
C A G C T G G T C G A C
SfiI  (2391)
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.
StuI  (2437)
1 site
A G G C C T T C C G G A
AvrII  (2438)
1 site
C C T A G G G G A T C C
HindIII  (2454)
1 site
A A G C T T T T C G A A
BspQI  (2569)
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  (2569)
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.
AflIII  (2685)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2685)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AlwNI  (3101)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
U6 forward sequencing primer
24-mer  /  38% GC
1 binding site
6486 .. 6509  =  24 annealed bases
Tm  =  55°C
AmpR
3505 .. 4365  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3505 .. 4296  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3505 .. 4365  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4297 .. 4365  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3505 .. 4365  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ZsGreen1
618 .. 1313  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
618 .. 1313  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ori
2746 .. 3334  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2746 .. 3334  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
3' LTR (ΔU3)
1433 .. 1858  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
3' LTR (ΔU3)
1433 .. 1858  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus
gag (truncated)
5878 .. 6294  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
5878 .. 6294  =  417 bp
truncated MMLV gag gene lacking the start codon
CMV enhancer
4656 .. 5035  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4656 .. 5035  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
2124 .. 2453  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2124 .. 2453  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
66 .. 369  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
66 .. 369  =  304 bp
human cytomegalovirus immediate early enhancer
U6 promoter
6318 .. 6558  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6318 .. 6558  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
370 .. 573  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
370 .. 573  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
5036 .. 5239  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
5036 .. 5239  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
MMLV Ψ
5478 .. 5677  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
MMLV Ψ
5478 .. 5677  =  200 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
5' LTR (truncated)
5240 .. 5415  =  176 bp
truncated long terminal repeat from Moloney murine
sarcoma virus
5' LTR (truncated)
5240 .. 5415  =  176 bp
truncated long terminal repeat from Moloney murine
sarcoma virus
AmpR promoter
4366 .. 4470  =  105 bp
AmpR promoter
4366 .. 4470  =  105 bp
SV40 ori
2304 .. 2439  =  136 bp
SV40 origin of replication
SV40 ori
2304 .. 2439  =  136 bp
SV40 origin of replication
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