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pQCXIH

Self-inactivating bicistronic retroviral vector for expression of a gene together with a hygromycin resistance marker.

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pQCXIH.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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SspI (7435) FspI (6853) AlwNI (6154) BspQI - SapI (5622) SfiI (5444) AccI (4610) SalI (4609) BmtI (4520) NheI (4516) PvuII (4417) EcoRV (4374) BsaBI * (4230) AleI (4165) BlpI (4065) AscI (542) BstEII (1252) XbaI (1595) 5' pQC Seq/PCR primer (2141 .. 2164) SbfI (2230) NotI (2243) AgeI (2250) BsiWI (2262) PacI (2273) BamHI (2278) 3' pQC Seq/PCR primer (2329 .. 2352) PspOMI (2421) ApaI (2425) PmlI (2624) AarI (2647) PflMI (2761) BmgBI (2851) AsiSI (3278) RsrII (3322) BbvCI (3979) pQCXIH 7780 bp
SspI  (7435)
1 site
A A T A T T T T A T A A
FspI  (6853)
1 site
T G C G C A A C G C G T
AlwNI  (6154)
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.
BspQI  (5622)
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  (5622)
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.
SfiI  (5444)
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.
AccI  (4610)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
SalI  (4609)
1 site
G T C G A C C A G C T G
BmtI  (4520)
1 site
G C T A G C C G A T C G
NheI  (4516)
1 site
G C T A G C C G A T C G
PvuII  (4417)
1 site
C A G C T G G T C G A C
EcoRV  (4374)
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.
BsaBI  (4230)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
AleI  (4165)
1 site
C A C N N N N G T G G T G N N N N C A C
BlpI  (4065)
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  (542)
1 site
G G C G C G C C C C G C G C G G
BstEII  (1252)
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.
XbaI  (1595)
1 site
T C T A G A A G A T C T
SbfI  (2230)
1 site
C C T G C A G G G G A C G T C C
NotI  (2243)
1 site
G C G G C C G C C G C C G G C G
AgeI  (2250)
1 site
A C C G G T T G G C C A
BsiWI  (2262)
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.
PacI  (2273)
1 site
T T A A T T A A A A T T A A T T
BamHI  (2278)
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.
PspOMI  (2421)
1 site
G G G C C C C C C G G G
ApaI  (2425)
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  (2624)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (2647)
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.
PflMI  (2761)
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.
BmgBI  (2851)
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.
AsiSI  (3278)
1 site
G C G A T C G C C G C T A G C G
RsrII  (3322)
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.
BbvCI  (3979)
1 site
C C T C A G C G G A G T C G
5' pQC Seq/PCR primer
24-mer  /  54% GC
1 binding site
2141 .. 2164  =  24 annealed bases
Tm  =  66°C
3' pQC Seq/PCR primer
24-mer  /  54% GC
1 binding site
2329 .. 2352  =  24 annealed bases
Tm  =  64°C
HygR
2920 .. 3939  =  1020 bp
339 amino acids  =  37.8 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
HygR
2920 .. 3939  =  1020 bp
339 amino acids  =  37.8 kDa
Product: aminoglycoside phosphotransferase from E. coli
confers resistance to hygromycin
AmpR
6558 .. 7418  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   6558 .. 7349  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6558 .. 7418  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7350 .. 7418  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6558 .. 7418  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
5799 .. 6387  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5799 .. 6387  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
2308 .. 2881  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
2308 .. 2881  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
3' LTR (ΔU3)
4486 .. 4911  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
3' LTR (ΔU3)
4486 .. 4911  =  426 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
gag (truncated)
1151 .. 1567  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
1151 .. 1567  =  417 bp
truncated MMLV gag gene lacking the start codon
CMV enhancer
7709 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
7709 .. 308  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5177 .. 5506  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5177 .. 5506  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
1604 .. 1907  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
1604 .. 1907  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
309 .. 512  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
309 .. 512  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
1908 .. 2111  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
1908 .. 2111  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
MMLV Ψ
751 .. 950  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
MMLV Ψ
751 .. 950  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
5' LTR (truncated)
513 .. 688  =  176 bp
truncated long terminal repeat from Moloney murine sarcoma virus
5' LTR (truncated)
513 .. 688  =  176 bp
truncated long terminal repeat from Moloney murine sarcoma virus
AmpR promoter
7419 .. 7523  =  105 bp
AmpR promoter
7419 .. 7523  =  105 bp
MCS
2225 .. 2283  =  59 bp
multiple cloning site
MCS
2225 .. 2283  =  59 bp
multiple cloning site
SV40 ori
5357 .. 5492  =  136 bp
SV40 origin of replication
SV40 ori
5357 .. 5492  =  136 bp
SV40 origin of replication
ORF:  2791 .. 3939  =  1149 bp
ORF:  382 amino acids  =  42.6 kDa
ORF:  6688 .. 6954  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3945 .. 4352  =  408 bp
ORF:  135 amino acids  =  14.3 kDa
ORF:  838 .. 1098  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  2806 .. 3672  =  867 bp
ORF:  288 amino acids  =  32.7 kDa
ORF:  4735 .. 5025  =  291 bp
ORF:  96 amino acids  =  10.5 kDa
ORF:  6558 .. 7418  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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