pKOV

Vector for knocking out E. coli genes using homologous recombination.

Sequence Author: Church Lab

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pKO3-R (4113 .. 4133) pKO3-L (996 .. 1016) NotI (1058) BamHI (4081) SalI (4093) pKOV 8662 bp
NotI  (1058)
1 site
G C G G C C G C C G C C G G C G
BamHI  (4081)
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.
SalI  (4093)
1 site
G T C G A C C A G C T G
pKO3-R
21-mer  /  62% GC
1 binding site
4113 .. 4133  =  21 annealed bases
Tm  =  65°C
sequencing primer
pKO3-L
21-mer  /  52% GC
1 binding site
996 .. 1016  =  21 annealed bases
Tm  =  59°C
sequencing primer
stuffer
1065 .. 4080  =  3016 bp
stuffer
1065 .. 4080  =  3016 bp
SacB
5115 .. 6536  =  1422 bp
473 amino acids  =  52.9 kDa
2 segments
   Segment 1:  signal peptide  
   5115 .. 5201  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
5115 .. 6536  =  1422 bp
473 amino acids  =  52.9 kDa
2 segments
   Segment 2:  
   5202 .. 6536  =  1335 bp
   444 amino acids  =  49.9 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
5115 .. 6536  =  1422 bp
473 amino acids  =  52.9 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
Rep101(Ts)
6929 .. 7879  =  951 bp
316 amino acids  =  37.4 kDa
Product: temperature-sensitive version of the RepA protein needed for replication with the pSC101 origin (Armstrong et al., 1984)
Rep101(Ts)
6929 .. 7879  =  951 bp
316 amino acids  =  37.4 kDa
Product: temperature-sensitive version of the RepA protein needed for replication with the pSC101 origin (Armstrong et al., 1984)
CmR
221 .. 880  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
221 .. 880  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
f1 ori
4111 .. 4565  =  455 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4111 .. 4565  =  455 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
sacB promoter
4669 .. 5114  =  446 bp
sacB promoter and control region
sacB promoter
4669 .. 5114  =  446 bp
sacB promoter and control region
pSC101 ori
7927 .. 8149  =  223 bp
low-copy replication origin that requires the Rep101 protein
pSC101 ori
7927 .. 8149  =  223 bp
low-copy replication origin that requires the Rep101 protein
cat promoter
118 .. 220  =  103 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
cat promoter
118 .. 220  =  103 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
ORF:  1213 .. 2061  =  849 bp
ORF:  282 amino acids  =  31.7 kDa
ORF:  221 .. 880  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  5115 .. 6536  =  1422 bp
ORF:  473 amino acids  =  52.9 kDa
ORF:  6929 .. 7879  =  951 bp
ORF:  316 amino acids  =  37.4 kDa
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Download pKOV.dna file

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