pSIREN-RetroQ-ZsGreen1

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

Sequence Author: Clontech (TaKaRa)

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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 enhancerSspI (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

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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
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
2 segments
   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
2 segments
   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
Truncated RNA polymerase III promoter for human U6 snRNA used in pSIREN vectors such as pSIREN-RetroQ-ZsGreen1. Expected transcription start site is 8bp downstream of this feature.
U6 promoter
6318 .. 6558  =  241 bp
Truncated RNA polymerase III promoter for human U6 snRNA used in pSIREN vectors such as pSIREN-RetroQ-ZsGreen1. Expected transcription start site is 8bp downstream of this feature.
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
ORF:  3635 .. 3901  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  618 .. 1313  =  696 bp
ORF:  231 amino acids  =  26.1 kDa
ORF:  5565 .. 5825  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  452 .. 718  =  267 bp
ORF:  88 amino acids  =  9.7 kDa
ORF:  995 .. 1288  =  294 bp
ORF:  97 amino acids  =  10.9 kDa
ORF:  1682 .. 1972  =  291 bp
ORF:  96 amino acids  =  10.5 kDa
ORF:  3505 .. 4365  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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