Gene replacement using pKO vectors

Description of pKO3

Excerpts from Andy Links 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 Qiagens 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&degree;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 Southerns.

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 notprimary sequence and has not been confirmed.Comments:Sequence file of the gene replacement vector pKO3. The vector has a temperature-sensitive pSC101 replicon whichwhich is functional at 30 C.  Hamiliton et. al. ,1989 J. Bacteriology 171: 4617-4622Link, 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 acetyltransferase1119-1575      f1 (+) filamentous origin2129-3550      Bacillus subtilis sacB gene4139-4149      oriC homologous region4204-4215      IHF binding site4407-5357      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 bpBased on pKO3: Link, A.J., Phillips, D. and Church, G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-typeEscherichia 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: 4617-4622NOTE: 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 beeasily used together with BamHI and because of a stuffer the double-digestcan be cleanly separated from singly cut contaminants by gel. The pKOV cloning site is: 5 - SmaI - NotI - SmaI- stuffer - BamHI - SalI - 3BamHI 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 andrelated galK promoter vectors are already in VectorDB:http://biology.queensu.ca/~miseners/vector_descrip/COMPLETE/PKO5.SEQ.htmlFor 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; region1048,1089    1048      SmaI cloning site(s)  1057     1057,1088   NotI cloning site(s) 1064-2456    none      PssR Left  flanking region2457-2579    none      c22 "universal" KO-tag  2580-3287    none      PssR right flanking region4079-4085  1068-1074   BamHI cloning site4091-4097  1099-1105   SalI cloning site 4115-4571  1119-1575   f1 (+) filamentous origin4658-6564  2129-3550   Bacillus subtilis sacB gene7131-8639  4139-4215   origin (pSC101)7399-8349  4407-5357   RepA (temperature-sensitive)     pkov  Length: 8673  May 9, 1999 04:58  Type: N  Check: 2716  ..CCCTTTCGTCTTCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTGCTACGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGAAAGCAAATTCGACCCGGTCGTCGGTTCAGGGCAGGGTCGTTAAATAGCCGCTTATGTCTATTGCTGGTTTANTCGGTACCCGGGGATCGCGGCCGCCCCGAAACCAACTGATGCAGTATCCCGGGCGCTACAACATGATCTCAGTGATGTTATCGGTCAGGAACAAGGAAAGCGAGGACTGGAAATTACCGCTGCTGGCGGGCACAACCTTTTACTGATTGGGCCGCCGGGAACAGGTAAAACAATGCTCGCCAGCCGTATTAATGGCCTGTTGCCAGATTTAAGCAATGAAGAGGCACTGGAGAGTGCTGCGATATTAAGTCTGGTAAATGCTGAATCAGTACAAAAACAATGGCGGCAGCGCCCGTTCCGCTCACCTCATCACAGTGCATCGTTAACTGCGATGGTAGGCGGTGGCGCAATTCCAGGGCCCGGTGAAATTTCGCTGGCGCATAACGGCGTGCTTTTTCTTGATGAGCTACCTGAATTTGAACGGCGTACACTGGATGCCTTGCGAGAGCCGATTGAATCCGGGCAGATCCATCTTTCACGCACACGAGCAAAAATAACCTATCCAGCCCGTTTCCAGCTTGTTGCGGCGATGAATCCCAGCCCTACCGGACATTATCAGGGAAACCATAACCGCTGCACGCCAGAACAGACATTACGTTATCTCAACCGGCTCTCGGGGCCCTTTCTCGACCGCTTCGATCTCTCACTGGAGATCCCATTACCACCCCCCGGCATTTTGAGTAAAACGGTAGTGCCGGGAGAAAGCAGCGCCACCGTTAAACAACGCGTAATGGCCGCCAGAGAGCGCCAATTTAAGCGGCAGAATAAACTGAATGCCTGGCTGGATAGTCCGGAAATACGCCAATTCTGCAAACTTGAGAGCGAAGATGCGATGTGGTTGGAAGGAACACTGATCCATCTGGGGTTATCGATTCGTGCCTGGCAGCGGTTATTGAAAGTTGCACGAACCATTGCTGATATTGATCAGTCTGACATTATCACACGTCAGCATTTGCAGGAGGCAGTTAGCTATCGAGCGATTGACCGTTTGCTCATCCATCTGCAGAAACTACTGACATAAAAAAAGGGCATTTCGCCCTTTTTATTAATCGTCAGAATCGGTGTAGTCTTCAGCACCTTCAACCTGCGGTTTACCGCGGAAAGGGTGTGAAAACGTTTTGGACGCTTAATACGCGTCATATACTTGGACCACACGCGTTCTGCTTCTGTCACTGGCTCACGTTCGCCACGGCATACTGCTACGAAGAGTTTCTCTTCCTCGGTAACCGGCTCGCGTTTGCCAAGATCCAACTCATTGAAGGCATAACCATGACGCTCAAGCAGTTGTGCCTCTTTGATGGTGAAATCACCATGACGAGAGAATCCACGTGGATAATGTTTATTGTCGAAATATCGATTAGTCGTCGTAAAGCTTTCCGCCATCCTGCACGCTCCTAATTCTTTGACCGAGCTAGTTATGGCGCGGAGTATTAGTTACGCTTGACAGAGTGTAAAACAAAACATTTAAATCATAACGACAAATAATTTTGCGGAGAGCACTGTGGATGTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTGGTAAAACAGCTGCATGAACGCCAGCTTGATCTTCTTATTACCACTGAAGCGCCCAAAATGGACGAATTTAGTAGTCAGTTGCTGGGATATTTCACTTTAGCGCTTTATACCAGTGCCCCTTCAAAACTAAAGGGAGATCTTAATTATCTGCGACTTGAGTGGGGGCCAGATTTTCAACAGCATGAGGCAGGTTTGATCGGTGCTGACGAAGTGCCCATTCTGACAACCAGTTCTGCTGAACTGGCACAGCAACAGATTGCGATGCTTAATGGTTGCACCTGGCTACCCGTCAGCTGGGCGCGTAAAAAAGGCGGCCTGCATACCGTTGTCGATAGCACAACACTTTCACGGCCGCTTTATGCCATATGGCTGCAAAATAGCGATAAAAATGCGTTGATTCGCGATCTTTTGAAAATTAACGTGCTGGATGAAGTGTATTAATATGAATGGCTGGCAAGGATGCCGGTAGAAGGATTTACTTCGGAGAGGGTTATTTCAGATAAAAAAAATCCTTACGTTTCGCTAAGGATGATTTCTGGCAGGGGCGGAGAGACTCGAACTCCCAACACCCGGTTTTGGAGACCGGTGCTCTACCAATTGAACTACGCCCCTAATTAGGGTGGCGGAACGGACGGGACTCGAACCCGCGACCCCCTGCGTGACAGGCAGGTATTCTAACCGACTGAACTACCGCTCCACCGAATTCTTTTACAACCACCGGTTTTATGACCGGCTTACTGCTTAATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACGGCCGCCAGGCAAATTCTGTTTCATCAGACCGCTTCTGCGTTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATCTTCGGCGTTGTAAGGTTAAGCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTGCGCTTACACACCCGGCCTATCAACGTCGTCGTCTTCAACGTTCCTTCAGGACTCTCAAGGAGTCAGGGAGAACTCATCTCGGGGCAAGTTTCGTGCTTAGATGCTTTCAGCACTTATCTCTTCCGCATTTAGCTACCGGGCAGTGCCATTGGCATGACAACCCGAACACCAGTGATGCGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCCCCTCAGTTCTCCAGCGCCCACGGCAGATAGGGACCGAACTGTCTCACGACGTTCTAAACCCAGCTCGCGTACCACTTTAAATGGCGAACAGCCATACCCTTGGGACCTACTTCAGCTCCAGGATGTGATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACTCTTGGGCGGTATCAGCCTGTTATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAACCACCGGATCACTATGACCTGCTTTCGCACCTGGGATCCTCTAGAGTCGACCGGNGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATNNCANGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTCCTGAATTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCAAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATNNCCGGTTTATTGACTACCGGAAGCAGTGTGACCGTGTGCTTCTCAAATGCCTCAGGCTGTCTATGTGTGACTGTTGAGCTGTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGTTTTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACATTGTCGATCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGTAAAAGCTCTGATGTATCTATCTTTTTTACACCGTTTTCATCTGTGCATATGGACAGTTTTCCCTTTGATATCTAACGGTGAACAGTTGTTCTACTTTTGTTTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCTCAGATCCTTCCGTATTTAGCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGCTTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAAAGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTGGTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCCTTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATCTCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCTCATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTAACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAACTTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCACAGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGATGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGGTTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGTTAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATCTCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTACTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACTTGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATATTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCCAGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAAGCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCTTTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACTACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACGGGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCAGCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACAGGCTTACCCGTCTTACTGTCNGGATCGACGCTCTCCCTTATGCGACTCCTGCAT

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