应用pKO3载体进行基因置换和基因敲除重组
产品名称： 应用pKO3载体进行基因置换和基因敲除重组
英文名称： Gene replacement using pKO vectors
产品编号： Biovector-T107923
产品价格： 5920
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使用范围： null
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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