Gene replacement using pKO vectors

Description of pKO3

Excerpts from Andy Link's 1994 Harvard University Thesis, "Experimental Tools for the Analysis of Genomes"

The vector pKO3 integrates into the chromosome by homologous recombination creating a tandem duplication at the nonpermissive temperature. When shifted to the permissive temperature, the pSC101 replication origin in the host chromosome is detrimental, and the vector is excised from the chromosome. To select for the loss of vector sequence from the cell, the B. subtilis gene sacB was incorporated into the vector, since expression of sacB in the presence of sucrose is detrimental to E. coli.

Because the entire protocol can be done by replica plating onto different selectable mediums at different temperatures, large numbers of genes can be simultaneously replaced with knockout alleles. Unlike other methods used for gene replacements in E. coli using ColE1 plasmids in a polA1 background (Guttersonand Koshland, 1983) or transformation of linear DNA into recBCsbcB or recD strains (Jasin and Schimmel, 1984; Winans et al., 1985; Shevell et al., 1988), this protocol is performed directly in wild-type strains. Moreover, since the system is plasmid based, gene replacements are easily performed in any genetic background that is recombination proficient.

Two approaches were developed for generating mutant alleles in vitro which could be inserted into the pKO3 vector for replacing open reading frames on the E. coli chromosome. Both methods were developed with the goal of eventually applying them to the large number of unidentified open reading frames. The first set of tools inserts an antibiotic marker into randomly cloned sequences to disrupt the open reading frame. From this first method, a second approach was derived which uses crossover PCR to generate precise deletions of the open reading frames. These two methods were applied to two E. coli reading frames identified in the protein content survey of E. coli.

Using pKO3

Andrew J. Link, Dereth R. Phillips, and George M. Church

Harvard Medical School

Department of Genetics

77 Avenue Louis Pasteur

Boston, MA 02115

The following methods were developed for working with the gene replacement vector pKO3. The plasmid and gene replacements protocols were derived from Hamilton et al. (1989) (J. Bacteriology 171: 4617-4622).

At the nonpermissive temperature, altered chromosomal sequences carried on the pSC101 plasmid integrates into the chromosome by homologous recombination to create an imperfect tandem duplication. When the cells are shifted to the permissive temperature, the cointegrant tends to undergoes a second recombination event regenerating the plasmid in the cell. Depending on the site of the second recombination, either the wild-type or the mutant allele is left behind in the chromosome. To select for the loss of vector sequence from the cell, the B. subtilis gene sacB was incorporated into the vector, since expression ofsacB in the presence of sucrose is detrimental to E. coli.

Strain: The gene replacement experiments used the recombination proficient strains EMG2 (F' lambda+) or MC1061 (F- araD139 del(araleu)7696 del(lacY74) galUgalKhsdrhsdM+ strA).

Media and growth conditions: All strains were grown in LB medium (1% (w/v) bactotryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl) with the appropriate selection. For antibiotic selection, the concentration of chromamphenicol was 20 mg/ml. For selection against sacB, LB medium was supplemented with sucrose to a final sucrose concentration of 5%(w/v)

NOTE: pKO3 has a temperature sensitive pSC101 replication origin. To recover the plasmid, strains harboring the plasmid must be grown at 30 deg C under chloramphenicol selection.

Isolation of pKO3: E. coli strains harboring pKO3 plasmid are grown in LB media at 30 deg C under chloramphenicol selection to stationary phase. Plasmid DNA is isolated usually the alkaline-lysis method (Birnboim and Doly) or Qiagen's plasmid prep kit.

For an analytical restriction digest of pKO3, 1.5 ml of overnight culture is used in the mini-prep, and the recovered plasmid DNA is resuspended in 25 ul of TE. 5 ul of the plasmid DNA is used for the restriction digestion and detection on ethidium bromide stained agarose gels. For preparing preparative amounts of pKO3, 50 to 1000 ml of overnight culture are used. The pSC101 plasmid is present at 5-10 copies per cell.

Electroporation: 40 ml of electroporation competent cells (1x10^11 cells/ml) were mixed with 1-3 ml of DNA in a ice-cold 500 ulmicrofuge tube and transferred to a 0.2 cm electroporationcuvette (Biorad, Inc). The cells are electroporated at 2,500 kV with 25 microfarad and 200 ohm resistance. Immediately after electroporation, 1 ml of SOC (2% bactotryptone, 0.5% yeast extract, 10 mMNaCl, 2.5 mMKCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) media is added to the cuvette. The cell are transferred to a 17 x 100 mm polypropylene tube and allowed to recover for 1 h at 30 deg C shaken at 250 rpm before plating on selective media.

DNA sequencing: Sequencing the left and right vector-insert junctions of inserts cloned into the BamHI site of pKO3 used the sequencing primers pK03-L and pK03-R. Cycle sequencing was performed essentially as described (Murray, 1989) using the Stratagene "Cyclist Sequencing" kit (Stratagene, Inc.) and plasmid DNA isolated using a Qiagen plasmid prep kit. Sequencing products were labeled with alpha-32P-dATP.

pK03-L: 5'-AGGGCAGGGTCGTTAAATAGC-3'

pK03-R: 5'-TTAATGCGCCGCTACAGGGCG-3'.

Polymerase chain reaction: Primers pK03-L and pK03-R were used for PCR amplifying inserts cloned into the BamHI site of pKO3. The PCR reactions used either purified DNA or bacterial colonies as the starting template. All PCR reactions were performed in a Perkin-Elmer 9600 thermal cycler. PCR reaction buffer (Ponce and Micol (1992), NAR 20: 623) consisted of 30 mMtricine (pH 8.4), 2 mM MgCl2, 5 mM beta-mercaptoethanol, 0.01% w/v gelatin, 0.1% w/v Thesit, 200 uM each dNTP, 600 nM of each primer, and 1 unit Taq polymerase (Boehringer Mannheim). The PCR reaction mixture was denatured at 94 deg C for 3 min before adding the Taq polymerase. The thermal cycle profile was 15 sec at 94 deg C, 15 sec at 55 deg C, and 30 sec at 72 deg C. All experiments used 30 cycles, and a final 5 min 72 deg C hold step.

Gene replacement: Mutant alleles cloned into the pKO3 gene replacement vector are electroporated into recombination proficient strains (eg. EMG2) and allowed to recover for 1 h at 30 deg C. The cells are plated on prewarmedchloramphenicol/LB plates and incubated at 42 deg C. To measure the integration frequency, the electroporated cells are also plated on chloramphenicol/LB plates at 30 deg C. From the 42 deg C plate, 1-5 colonies are picked into 1 ml of LB broth, serially diluted, and immediately plated at 30°ree;C on either 5% w/v sucrose or 5% sucrose+antibiotic plates. The 5% sucrose plates are replica plated to chloramphenicol plates at 30 deg C to test for loss of the replacement vector (cms). The gene replacement is confirmed by either PCR using primers flanking the targeted open reading frame or by genomic Southern's.

IMPORTANT THINGS TO REMEMBER:

1)    pK03 has a temperature sensitive Psc101 replication orgin.  Strains harboring the plasmid must be grown at 30 deg C under chloramphenicol selection.

2)    SacB appears to be mildly stressful to the cell even without added sucrose.  It is a good idea not to carry the plasmid for too many generations in a single strain as you will accumulate mutations either in the sacB gene or in the bacterial genome to bypass this toxicity.

3)    Make sure you test your plasmid for all of the markers (ts, sacB, cat) before you clone anything into it.  We generally check each batch of pK03 before shipping.  In the absence of selection the ts and sacB markers are basically stable.  The pK03 strain has "very-slow-growth" (NOT "no-growth") at 43 deg or on sucrose, so streak tests are not recommended.  Instead, we take a pK03 "test-colony" and serially dilute and plate for single colonies at both 30 and 40 degrees.  If the 43 degree colonies are smaller than the 30 degree colonies then the "test-colony" is "ts".  When performing the knock-out protocol, we only pick the large colonies on the 43 deg plates.  We do the same serial dilution with sucrose/LB versus LB plates to test for SacB.

4)    We are not prepared to advise researchers on the use of this plasmid in any other species or using any cloning sites other than those used in the J. Bact. Paper.  All other uses are at you won risk.

5)    The pSC101 plasmid is considered to be low copy and is present at 5-10 copies per cell. You may need to adjust your plasmid prep protocols to increase your yield.

For additional updates, vector and tag sequences and information about E. coli community gene knockout resource sharing please consult our web site: http://arep.med.harvard.edu/gmc/ecoko.html

pKO3 & pKOV Maps and Sequence

Figure 1

A map of pKO3 showing the unique restriction sites. The sequences \used to construct the map have not been verified. With the exception of BamHI, NotI, and HindIII, the restriction sites have not been confirmed.

Figure 2

A detailed map of the BamHI cloning site used for cloning genomic inserts for gene replacement experiments in the Church lab. pK03-L and pK03-R are primer sequences used for PCR amplification across the cloning site and cycle sequencing the vector-insert junctions.

Sequence files (GCG format):

Gene replacement using the pKO3 vector

   
NOTE: The file is constructed from various GenBank files and is not
primary sequence and has not been confirmed.

Comments:
Sequence file of the gene replacement vector pKO3. 
The vector has a temperature-sensitive pSC101 replicon which
which is functional at 30 C.  
Hamiliton et. al. ,1989 J. Bacteriology 171: 4617-4622

Link, A.J., Phillips, D. and Church, G.M. (1997) 
Methods for generating precise deletions and insertions in the 
genome of wild-type Escherichia coli: 
Application to open reading frame characterization. 
J. Bacteriology 179: 6228-6237. 

Current base coordinates:
221-880        Cm-R; chloramphenicol acetyltransferase
1119-1575      f1 (+) filamentous origin
2129-3550      Bacillus subtilis sacB gene
4139-4149      oriC homologous region
4204-4215      IHF binding site
4407-5357      RepA (temperature-sensitive ?) 
Pko3.Mail  Length: 5681  May 31, 1995  11:25  Type: N  Check: 5438  ..

       1  CCCTTTCGTC TTCGAATAAA TACCTGTGAC GGAAGATCAC TTCGCAGAAT 
      51  AAATAAATCC TGGTGTCCCT GTTGATACCG GGAAGCCCTG GGCCAACTTT 
     101  TGGCGAAAAT GAGACGTTGA TCGGCACGTA AGAGGTTCCA ACTTTCACCA 
     151  TAATGAAATA AGATCACTAC CGGGCGTATT TTTTGAGTTA TCGAGATTTT 
     201  CAGGAGCTAA GGAAGCTAAA ATGGAGAAAA AAATCACTGG ATATACCACC 
     251  GTTGATATAT CCCAATGGCA TCGTAAAGAA CATTTTGAGG CATTTCAGTC 
     301  AGTTGCTCAA TGTACCTATA ACCAGACCGT TCAGCTGGAT ATTACGGCCT 
     351  TTTTAAAGAC CGTAAAGAAA AATAAGCACA AGTTTTATCC GGCCTTTATT 
     401  CACATTCTTG CCCGCCTGAT GAATGCTCAT CCGGAATTCC GTATGGCAAT 
     451  GAAAGACGGT GAGCTGGTGA TATGGGATAG TGTTCACCCT TGTTACACCG 
     501  TTTTCCATGA GCAAACTGAA ACGTTTTCAT CGCTCTGGAG TGAATACCAC 
     551  GACGATTTCC GGCAGTTTCT ACACATATAT TCGCAAGATG TGGCGTGTTA 
     601  CGGTGAAAAC CTGGCCTATT TCCCTAAAGG GTTTATTGAG AATATGTTTT 
     651  TCGTCTCAGC CAATCCCTGG GTGAGTTTCA CCAGTTTTGA TTTAAACGTG 
     701  GCCAATATGG ACAACTTCTT CGCCCCCGTT TTCACCATGG GCAAATATTA 
     751  TACGCAAGGC GACAAGGTGC TGATGCCGCT GGCGATTCAG GTTCATCATG 
     801  CCGTTTGTGA TGGCTTCCAT GTCGGCAGAA TGCTTAATGA ATTACAACAG 
     851  TACTGCGATG AGTGGCAGGG CGGGGCGTAA TTTTTTTAAG GCAGTTATTG 
     901  GTGCCCTTAA ACGCCTGGTG CTACGCCTGA ATAAGTGATA ATAAGCGGAT 
     951  GAATGGCAGA AATTCGAAAG CAAATTCGAC CCGGTCGTCG GTTCAGGGCA 
    1001  GGGTCGTTAA ATAGCCGCTT ATGTCTATTG CTGGTTTANT CGGTACCCGG 
    1051  Ggatcgcggc cgcggaccgg atcctctaga gcggccgcGA TCCTCTAGAG 
    1101  TCGACCGGNG AATGGCGAAT GGGACGCGCC CTGTAGCGGC GCATTAAGCG 
    1151  CGGCGGGTGT GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC 
    1201  CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC 
    1251  CGGCTTTCCC CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT 
    1301  TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT 
    1351  TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT 
    1401  GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC 
    1451  TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT 
    1501  TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA 
    1551  TTTTAACAAA ATATTAACGC TTACAATTTA GGTGGCACTT TTCGGGGAAA 
    1601  TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT 
    1651  ATCCGCTCAT NNCANGATCC TTTTTAACCC ATCACATATA CCTGCCGTTC 
    1701  ACTATTATTT AGTGAAATGA GATATTATGA TATTTTCTGA ATTGTGATTA 
    1751  AAAAGGCAAC TTTATGCCCA TGCAACAGAA ACTATAAAAA ATACAGAGAA 
    1801  TGAAAAGAAA CAGATAGATT TTTTAGTTCT TTAGGCCCGT AGTCTGCAAA 
    1851  TCCTTTTATG ATTTTCTATC AAACAAAAGA GGAAAATAGA CCAGTTGCAA 
    1901  TCCAAACGAG AGTCTAATAG AATGAGGTCG AAAAGTAAAT CGCGCGGGTT 
    1951  TGTTACTGAT AAAGCAGGCA AGACCTAAAA TGTGTAAAGG GCAAAGTGTA 
    2001  TACTTTGGCG TCACCCCTTA CATATTTTAG GTCTTTTTTT ATTGTGCGTA 
    2051  ACTAACTTGC CATCTTCAAA CAGGAGGGCT GGAAGAAGCA GACCGCTAAC 
    2101  ACAGTACATA AAAAAGGAGA CATGAACGAT GAACATCAAA AAGTTTGCAA 
    2151  AACAAGCAAC AGTATTAACC TTTACTACCG CACTGCTGGC AGGAGGCGCA 
    2201  ACTCAAGCGT TTGCGAAAGA AACGAACCAA AAGCCATATA AGGAAACATA 
    2251  CGGCATTTCC CATATTACAC GCCATGATAT GCTGCAAATC CCTGAACAGC 
    2301  AAAAAAATGA AAAATATCAA GTTCCTGAAT TCGATTCGTC CACAATTAAA 
    2351  AATATCTCTT CTGCAAAAGG CCTGGACGTT TGGGACAGCT GGCCATTACA 
    2401  AAACGCTGAC GGCACTGTCG CAAACTATCA CGGCTACCAC ATCGTCTTTG 
    2451  CATTAGCCGG AGATCCTAAA AATGCGGATG ACACATCGAT TTACATGTTC 
    2501  TATCAAAAAG TCGGCGAAAC TTCTATTGAC AGCTGGAAAA ACGCTGGCCG 
    2551  CGTCTTTAAA GACAGCGACA AATTCGATGC AAATGATTCT ATCCTAAAAG 
    2601  ACCAAACACA AGAATGGTCA GGTTCAGCCA CATTTACATC TGACGGAAAA 
    2651  ATCCGTTTAT TCTACACTGA TTTCTCCGGT AAACATTACG GCAAACAAAC 
    2701  ACTGACAACT GCACAAGTTA ACGTATCAGC ATCAGACAGC TCTTTGAACA 
    2751  TCAACGGTGT AGAGGATTAT AAATCAATCT TTGACGGTGA CGGAAAAACG 
    2801  TATCAAAATG TACAGCAGTT CATCGATGAA GGCAACTACA GCTCAGGCGA 
    2851  CAACCATACG CTGAGAGATC CTCACTACGT AGAAGATAAA GGCCACAAAT 
    2901  ACTTAGTATT TGAAGCAAAC ACTGGAACTG AAGATGGCTA CCAAGGCGAA 
    2951  GAATCTTTAT TTAACAAAGC ATACTATGGC AAAAGCACAT CATTCTTCCG 
    3001  TCAAGAAAGT CAAAAACTTC TGCAAAGCGA TAAAAAACGC ACGGCTGAGT 
    3051  TAGCAAACGG CGCTCTCGGT ATGATTGAGC TAAACGATGA TTACACACTG 
    3101  AAAAAAGTGA TGAAACCGCT GATTGCATCT AACACAGTAA CAGATGAAAT 
    3151  TGAACGCGCG AACGTCTTTA AAATGAACGG CAAATGGTAC CTGTTCACTG 
    3201  ACTCCCGCGG ATCAAAAATG ACGATTGACG GCATTACGTC TAACGATATT 
    3251  TACATGCTTG GTTATGTTTC TAATTCTTTA ACTGGCCCAT ACAAGCCGCT 
    3301  GAACAAAACT GGCCTTGTGT TAAAAATGGA TCTTGATCCT AACGATGTAA 
    3351  CCTTTACTTA CTCACACTTC GCTGTACCTC AAGCGAAAGG AAACAATGTC 
    3401  GTGATTACAA GCTATATGAC AAACAGAGGA TTCTACGCAG ACAAACAATC 
    3451  AACGTTTGCG CCAAGCTTCC TGCTGAACAT CAAAGGCAAG AAAACATCTG 
    3501  TTGTCAAAGA CAGCATCCTT GAACAAGGAC AATTAACAGT TAACAAATAA 
    3551  AAACGCAAAA GAAAATGCCG ATNNCCGGTT TATTGACTAC CGGAAGCAGT 
    3601  GTGACCGTGT GCTTCTCAAA TGCCTCAGGC TGTCTATGTG TGACTGTTGA 
    3651  GCTGTAACAA GTTGTCTCAG GTGTTCAATT TCATGTTCTA GTTGCTTTGT 
    3701  TTTACTGGTT TCACCTGTTC TATTAGGTGT TACATGCTGT TCATCTGTTA 
    3751  CATTGTCGAT CTGTTCATGG TGAACAGCTT TAAATGCACC AAAAACTCGT 
    3801  AAAAGCTCTG ATGTATCTAT CTTTTTTACA CCGTTTTCAT CTGTGCATAT 
    3851  GGACAGTTTT CCCTTTGATA TCTAACGGTG AACAGTTGTT CTACTTTTGT 
    3901  TTGTTAGTCT TGATGCTTCA CTGATAGATA CAAGAGCCAT AAGAACCTCA 
    3951  GATCCTTCCG TATTTAGCCA GTATGTTCTC TAGTGTGGTT CGTTGTTTTT 
    4001  GCGTGAGCCA TGAGAACGAA CCATTGAGAT CATGCTTACT TTGCATGTCA 
    4051  CTCAAAAATT TTGCCTCAAA ACTGGTGAGC TGAATTTTTG CAGTTAAAGC 
    4101  ATCGTGTAGT GTTTTTCTTA GTCCGTTACG TAGGTAGGAA TCTGATGTAA 
    4151  TGGTTGTTGG TATTTTGTCA CCATTCATTT TTATCTGGTT GTTCTCAAGT 
    4201  TCGGTTACGA GATCCATTTG TCTATCTAGT TCAACTTGGA AAATCAACGT 
    4251  ATCAGTCGGG CGGCCTCGCT TATCAACCAC CAATTTCATA TTGCTGTAAG 
    4301  TGTTTAAATC TTTACTTATT GGTTTCAAAA CCCATTGGTT AAGCCTTTTA 
    4351  AACTCATGGT AGTTATTTTC AAGCATTAAC ATGAACTTAA ATTCATCAAG 
    4401  GCTAATCTCT ATATTTGCCT TGTGAGTTTT CTTTTGTGTT AGTTCTTTTA 
    4451  ATAACCACTC ATAAATCCTC ATAGAGTATT TGTTTTCAAA AGACTTAACA 
    4501  TGTTCCAGAT TATATTTTAT GAATTTTTTT AACTGGAAAA GATAAGGCAA 
    4551  TATCTCTTCA CTAAAAACTA ATTCTAATTT TTCGCTTGAG AACTTGGCAT 
    4601  AGTTTGTCCA CTGGAAAATC TCAAAGCCTT TAACCAAAGG ATTCCTGATT 
    4651  TCCACAGTTC TCGTCATCAG CTCTCTGGTT GCTTTAGCTA ATACACCATA 
    4701  AGCATTTTCC CTACTGATGT TCATCATCTG AGCGTATTGG TTATAAGTGA 
    4751  ACGATACCGT CCGTTCTTTC CTTGTAGGGT TTTCAATCGT GGGGTTGAGT 
    4801  AGTGCCACAC AGCATAAAAT TAGCTTGGTT TCATGCTCCG TTAAGTCATA 
    4851  GCGACTAATC GCTAGTTCAT TTGCTTTGAA AACAACTAAT TCAGACATAC 
    4901  ATCTCAATTG GTCTAGGTGA TTTTAATCAC TATACCAATT GAGATGGGCT 
    4951  AGTCAATGAT AATTACTAGT CCTTTTCCTT TGAGTTGTGG GTATCTGTAA 
    5001  ATTCTGCTAG ACCTTTGCTG GAAAACTTGT AAATTCTGCT AGACCCTCTG 
    5051  TAAATTCCGC TAGACCTTTG TGTGTTTTTT TTGTTTATAT TCAAGTGGTT 
    5101  ATAATTTATA GAATAAAGAA AGAATAAAAA AAGATAAAAA GAATAGATCC 
    5151  CAGCCCTGTG TATAACTCAC TACTTTAGTC AGTTCCGCAG TATTACAAAA 
    5201  GGATGTCGCA AACGCTGTTT GCTCCTCTAC AAAACAGACC TTAAAACCCT 
    5251  AAAGGCTTAA GTAGCACCCT CGCAAGCTCG GGCAAATCGC TGAATATTCC 
    5301  TTTTGTCTCC GACCATCAGG CACCTGAGTC GCTGTCTTTT TCGTGACATT 
    5351  CAGTTCGCTG CGCTCACGGC TCTGGCAGTG AATGGGGGTA AATGGCACTA 
    5401  CAGGCGCCTT TTATGGATTC ATGCAAGGAA ACTACCCATA ATACAAGAAA 
    5451  AGCCCGTCAC GGGCTTCTCA GGGCGTTTTA TGGCGGGTCT GCTATGTGGT 
    5501  GCTATCTGAC TTTTTGCTGT TCAGCAGTTC CTGCCCTCTG ATTTTCCAGT 
    5551  CTGACCACTT CGGATTATCC CGTGACAGGT CATTCAGACT GGCTAATGCA 
    5601  CCCAGTAAGG CAGCGGTATC ATCAACAGGC TTACCCGTCT TACTGTCnGG 
    5651  ATCGACGCTC TCCCTTATGC GACTCCTGCA T

PKOV  (pKO5)  16-Jan-1996    8673 bp

Based on pKO3: Link, A.J., Phillips, D. and Church, G.M. (1997) Methods 
for generating precise deletions and insertions in the genome of wild-type
Escherichia coli: Application to open reading frame 
characterization. J. Bacteriology 179: 6228-6237. 
The temperature-sensitive pSC101 replicon is functional at 30 C; 
not at 43 C; Hamilton et. al. (1989) J. Bacteriology 171: 4617-4622

NOTE: This file is constructed from various GenBank files and is not 
primary sequence and has not been confirmed.

PKOV has slight advantages over pKO3 in that the NotI cloning site can be
easily used together with BamHI and because of a stuffer the double-digest
can be cleanly separated from singly cut contaminants by gel. The pKOV 
cloning site is: 5' - SmaI - NotI - SmaI- stuffer - BamHI - SalI - 3'
BamHI and SalI are not used together, nor is SmaI used with NotI. BglII & 
BclI cut ends are compatible with BamHI. PmeI & SwaI are compatible with SmaI.

pKOV was previously named pKO5, but has changed name since pKO5 and
related galK promoter vectors are already in VectorDB:
http://biology.queensu.ca/~miseners/vector_descrip/COMPLETE/PKO5.SEQ.html

For applications and updates see: http://arep.med.harvard.edu      
                                  ***************************

   pKOV     pKO3   base coordinates :
 221- 880   221- 880   Cm-R; chloramphenicol acetyltransferase
  13-1038    13-1038   Cm-R; region
1048,1089    1048      SmaI cloning site(s)
  1057     1057,1088   NotI cloning site(s) 
1064-2456    none      PssR Left  flanking region
2457-2579    none      c22 "universal" KO-tag  
2580-3287    none      PssR right flanking region
4079-4085  1068-1074   BamHI cloning site
4091-4097  1099-1105   SalI cloning site 
4115-4571  1119-1575   f1 (+) filamentous origin
4658-6564  2129-3550   Bacillus subtilis sacB gene
7131-8639  4139-4215   origin (pSC101)
7399-8349  4407-5357   RepA (temperature-sensitive)

     pkov  Length: 8673  May 9, 1999 04:58  Type: N  Check: 2716  ..

CCCTTTCGTCTTCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTG
TTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAAC
TTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGA
AGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATT
TTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTA
AAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGC
TCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACA
CCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTT
CTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGA
GAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGG
ACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTG
GCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTA
CTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTGCTA
CGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGAAAGCAAATTCGACCCGGTCGTCGGTTC
AGGGCAGGGTCGTTAAATAGCCGCTTATGTCTATTGCTGGTTTANTCGGTACCCGGGGATCGCGGCCGCCC
CGAAACCAACTGATGCAGTATCCCGGGCGCTACAACATGATCTCAGTGATGTTATCGGTCAGGAACAAGGA
AAGCGAGGACTGGAAATTACCGCTGCTGGCGGGCACAACCTTTTACTGATTGGGCCGCCGGGAACAGGTAA
AACAATGCTCGCCAGCCGTATTAATGGCCTGTTGCCAGATTTAAGCAATGAAGAGGCACTGGAGAGTGCTG
CGATATTAAGTCTGGTAAATGCTGAATCAGTACAAAAACAATGGCGGCAGCGCCCGTTCCGCTCACCTCAT
CACAGTGCATCGTTAACTGCGATGGTAGGCGGTGGCGCAATTCCAGGGCCCGGTGAAATTTCGCTGGCGCA
TAACGGCGTGCTTTTTCTTGATGAGCTACCTGAATTTGAACGGCGTACACTGGATGCCTTGCGAGAGCCGA
TTGAATCCGGGCAGATCCATCTTTCACGCACACGAGCAAAAATAACCTATCCAGCCCGTTTCCAGCTTGTT
GCGGCGATGAATCCCAGCCCTACCGGACATTATCAGGGAAACCATAACCGCTGCACGCCAGAACAGACATT
ACGTTATCTCAACCGGCTCTCGGGGCCCTTTCTCGACCGCTTCGATCTCTCACTGGAGATCCCATTACCAC
CCCCCGGCATTTTGAGTAAAACGGTAGTGCCGGGAGAAAGCAGCGCCACCGTTAAACAACGCGTAATGGCC
GCCAGAGAGCGCCAATTTAAGCGGCAGAATAAACTGAATGCCTGGCTGGATAGTCCGGAAATACGCCAATT
CTGCAAACTTGAGAGCGAAGATGCGATGTGGTTGGAAGGAACACTGATCCATCTGGGGTTATCGATTCGTG
CCTGGCAGCGGTTATTGAAAGTTGCACGAACCATTGCTGATATTGATCAGTCTGACATTATCACACGTCAG
CATTTGCAGGAGGCAGTTAGCTATCGAGCGATTGACCGTTTGCTCATCCATCTGCAGAAACTACTGACATA
AAAAAAGGGCATTTCGCCCTTTTTATTAATCGTCAGAATCGGTGTAGTCTTCAGCACCTTCAACCTGCGGT
TTACCGCGGAAAGGGTGTGAAAACGTTTTGGACGCTTAATACGCGTCATATACTTGGACCACACGCGTTCT
GCTTCTGTCACTGGCTCACGTTCGCCACGGCATACTGCTACGAAGAGTTTCTCTTCCTCGGTAACCGGCTC
GCGTTTGCCAAGATCCAACTCATTGAAGGCATAACCATGACGCTCAAGCAGTTGTGCCTCTTTGATGGTGA
AATCACCATGACGAGAGAATCCACGTGGATAATGTTTATTGTCGAAATATCGATTAGTCGTCGTAAAGCTT
TCCGCCATCCTGCACGCTCCTAATTCTTTGACCGAGCTAGTTATGGCGCGGAGTATTAGTTACGCTTGACA
GAGTGTAAAACAAAACATTTAAATCATAACGACAAATAATTTTGCGGAGAGCACTGTGGATGTTATAAATT
TGGAGTGTGAAGGTTATTGCGTGTGGTAAAACAGCTGCATGAACGCCAGCTTGATCTTCTTATTACCACTG
AAGCGCCCAAAATGGACGAATTTAGTAGTCAGTTGCTGGGATATTTCACTTTAGCGCTTTATACCAGTGCC
CCTTCAAAACTAAAGGGAGATCTTAATTATCTGCGACTTGAGTGGGGGCCAGATTTTCAACAGCATGAGGC
AGGTTTGATCGGTGCTGACGAAGTGCCCATTCTGACAACCAGTTCTGCTGAACTGGCACAGCAACAGATTG
CGATGCTTAATGGTTGCACCTGGCTACCCGTCAGCTGGGCGCGTAAAAAAGGCGGCCTGCATACCGTTGTC
GATAGCACAACACTTTCACGGCCGCTTTATGCCATATGGCTGCAAAATAGCGATAAAAATGCGTTGATTCG
CGATCTTTTGAAAATTAACGTGCTGGATGAAGTGTATTAATATGAATGGCTGGCAAGGATGCCGGTAGAAG
GATTTACTTCGGAGAGGGTTATTTCAGATAAAAAAAATCCTTACGTTTCGCTAAGGATGATTTCTGGCAGG
GGCGGAGAGACTCGAACTCCCAACACCCGGTTTTGGAGACCGGTGCTCTACCAATTGAACTACGCCCCTAA
TTAGGGTGGCGGAACGGACGGGACTCGAACCCGCGACCCCCTGCGTGACAGGCAGGTATTCTAACCGACTG
AACTACCGCTCCACCGAATTCTTTTACAACCACCGGTTTTATGACCGGCTTACTGCTTAATTTGATGCCTG
GCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCG
GCATGGGGTCAGGTGGGACCACCGCGCTACGGCCGCCAGGCAAATTCTGTTTCATCAGACCGCTTCTGCGT
TCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATCTTCGGCGTTGTAAGGTTAA
GCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTGCGCTTACACACCCGGCCTATCAACGTCGT
CGTCTTCAACGTTCCTTCAGGACTCTCAAGGAGTCAGGGAGAACTCATCTCGGGGCAAGTTTCGTGCTTAG
ATGCTTTCAGCACTTATCTCTTCCGCATTTAGCTACCGGGCAGTGCCATTGGCATGACAACCCGAACACCA
GTGATGCGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCCCCTCAGTTCTCCAGCGCCCACGGCAGATA
GGGACCGAACTGTCTCACGACGTTCTAAACCCAGCTCGCGTACCACTTTAAATGGCGAACAGCCATACCCT
TGGGACCTACTTCAGCTCCAGGATGTGATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACT
CTTGGGCGGTATCAGCCTGTTATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAAC
CACCGGATCACTATGACCTGCTTTCGCACCTGGGATCCTCTAGAGTCGACCGGNGAATGGCGAATGGGACG
CGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC
GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGC
TCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATT
AGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACG
TTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT
ATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT
TTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTT
TATTTTTCTAAATACATTCAAATATGTATCCGCTCATNNCANGATCCTTTTTAACCCATCACATATACCTG
CCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTAT
GCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGC
CCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAAC
GAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAA
AATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGT
AACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGA
CATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCA
GGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCA
TATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTCCTGAATTCG
ATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAAC
GCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGC
GGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTG
GCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGG
TCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTA
CGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTG
TAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAA
GGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATA
CTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCAT
ACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACG
GCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAA
ACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAAT
GGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATG
CTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAAT
GGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATG
TCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCAAGCTTC
CTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGT
TAACAAATAAAAACGCAAAAGAAAATGCCGATNNCCGGTTTATTGACTACCGGAAGCAGTGTGACCGTGTG
CTTCTCAAATGCCTCAGGCTGTCTATGTGTGACTGTTGAGCTGTAACAAGTTGTCTCAGGTGTTCAATTTC
ATGTTCTAGTTGCTTTGTTTTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACAT
TGTCGATCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGTAAAAGCTCTGATGTATCTATCTTT
TTTACACCGTTTTCATCTGTGCATATGGACAGTTTTCCCTTTGATATCTAACGGTGAACAGTTGTTCTACT
TTTGTTTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCTCAGATCCTTCCGTATTTA
GCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGCTT
ACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAAAGCATCGTGTA
GTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTGGTATTTTGTCACCATTCATT
TTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATCAACGT
ATCAGTCGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTG
GTTTCAAAACCCATTGGTTAAGCCTTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAAT
TCATCAAGGCTAATCTCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATA
AATCCTCATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTAACT
GGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAACTTGGCATAGTTT
GTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCACAGTTCTCGTCATCAGCTCTCT
GGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGATGTTCATCATCTGAGCGTATTGGTTATAAG
TGAACGATACCGTCCGTTCTTTCCTTGTAGGGTTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAA
ATTAGCTTGGTTTCATGCTCCGTTAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAA
TTCAGACATACATCTCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGAT
AATTACTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACTTGTA
AATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATATTCAAGTGGTTAT
AATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCCAGCCCTGTGTATAACTCACTAC
TTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAGACCTTAA
AACCCTAAAGGCTTAAGTAGCACCCTCGCAAGCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCA
TCAGGCACCTGAGTCGCTGTCTTTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGG
GGTAAATGGCACTACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGT
CACGGGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCAGCAGT
TCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGACTGGCTAATGC
ACCCAGTAAGGCAGCGGTATCATCAACAGGCTTACCCGTCTTACTGTCNGGATCGACGCTCTCCCTTATGC
GACTCCTGCAT

These files have been constructed from various GenBank files, are not primary data, and have not been confirmed.