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°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 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
![](\"http://img.ebiomall.cn/images/sell/547fdb2631e91.jpg\" "\\"pKO3")
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
![](\"http://img.ebiomall.cn/images/sell/547fdb281a46d.jpg\" "\\"pKO3_CLONING_SITE,")
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:221-880Cm-R; chloramphenicol acetyltransferase1119-1575f1 (+) filamentous origin2129-3550Bacillus subtilis sacB gene4139-4149oriC homologous region4204-4215IHF binding site4407-5357RepA (temperature-sensitive ?) Pko3.MailLength: 5681May 31, 199511:25Type: NCheck: 5438.. 1CCCTTTCGTC TTCGAATAAA TACCTGTGAC GGAAGATCAC TTCGCAGAAT 51AAATAAATCC TGGTGTCCCT GTTGATACCG GGAAGCCCTG GGCCAACTTT101TGGCGAAAAT GAGACGTTGA TCGGCACGTA AGAGGTTCCA ACTTTCACCA151TAATGAAATA AGATCACTAC CGGGCGTATT TTTTGAGTTA TCGAGATTTT201CAGGAGCTAA GGAAGCTAAA ATGGAGAAAA AAATCACTGG ATATACCACC251GTTGATATAT CCCAATGGCA TCGTAAAGAA CATTTTGAGG CATTTCAGTC301AGTTGCTCAA TGTACCTATA ACCAGACCGT TCAGCTGGAT ATTACGGCCT351TTTTAAAGAC CGTAAAGAAA AATAAGCACA AGTTTTATCC GGCCTTTATT401CACATTCTTG CCCGCCTGAT GAATGCTCAT CCGGAATTCC GTATGGCAAT451GAAAGACGGT GAGCTGGTGA TATGGGATAG TGTTCACCCT TGTTACACCG501TTTTCCATGA GCAAACTGAA ACGTTTTCAT CGCTCTGGAG TGAATACCAC551GACGATTTCC GGCAGTTTCT ACACATATAT TCGCAAGATG TGGCGTGTTA601CGGTGAAAAC CTGGCCTATT TCCCTAAAGG GTTTATTGAG AATATGTTTT651TCGTCTCAGC CAATCCCTGG GTGAGTTTCA CCAGTTTTGA TTTAAACGTG701GCCAATATGG ACAACTTCTT CGCCCCCGTT TTCACCATGG GCAAATATTA751TACGCAAGGC GACAAGGTGC TGATGCCGCT GGCGATTCAG GTTCATCATG801CCGTTTGTGA TGGCTTCCAT GTCGGCAGAA TGCTTAATGA ATTACAACAG851TACTGCGATG AGTGGCAGGG CGGGGCGTAA TTTTTTTAAG GCAGTTATTG901GTGCCCTTAA ACGCCTGGTG CTACGCCTGA ATAAGTGATA ATAAGCGGAT951GAATGGCAGA AATTCGAAAG CAAATTCGAC CCGGTCGTCG GTTCAGGGCA 1001GGGTCGTTAA ATAGCCGCTT ATGTCTATTG CTGGTTTANT CGGTACCCGG 1051Ggatcgcggc cgcggaccgg atcctctaga gcggccgcGA TCCTCTAGAG 1101TCGACCGGNG AATGGCGAAT GGGACGCGCC CTGTAGCGGC GCATTAAGCG 1151CGGCGGGTGT GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC 1201CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC 1251CGGCTTTCCC CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT 1301TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT 1351TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT 1401GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC 1451TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT 1501TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA 1551TTTTAACAAA ATATTAACGC TTACAATTTA GGTGGCACTT TTCGGGGAAA 1601TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT 1651ATCCGCTCAT NNCANGATCC TTTTTAACCC ATCACATATA CCTGCCGTTC 1701ACTATTATTT AGTGAAATGA GATATTATGA TATTTTCTGA ATTGTGATTA 1751AAAAGGCAAC TTTATGCCCA TGCAACAGAA ACTATAAAAA ATACAGAGAA 1801TGAAAAGAAA CAGATAGATT TTTTAGTTCT TTAGGCCCGT AGTCTGCAAA 1851TCCTTTTATG ATTTTCTATC AAACAAAAGA GGAAAATAGA CCAGTTGCAA 1901TCCAAACGAG AGTCTAATAG AATGAGGTCG AAAAGTAAAT CGCGCGGGTT 1951TGTTACTGAT AAAGCAGGCA AGACCTAAAA TGTGTAAAGG GCAAAGTGTA 2001TACTTTGGCG TCACCCCTTA CATATTTTAG GTCTTTTTTT ATTGTGCGTA 2051ACTAACTTGC CATCTTCAAA CAGGAGGGCT GGAAGAAGCA GACCGCTAAC 2101ACAGTACATA AAAAAGGAGA CATGAACGAT GAACATCAAA AAGTTTGCAA 2151AACAAGCAAC AGTATTAACC TTTACTACCG CACTGCTGGC AGGAGGCGCA 2201ACTCAAGCGT TTGCGAAAGA AACGAACCAA AAGCCATATA AGGAAACATA 2251CGGCATTTCC CATATTACAC GCCATGATAT GCTGCAAATC CCTGAACAGC 2301AAAAAAATGA AAAATATCAA GTTCCTGAAT TCGATTCGTC CACAATTAAA 2351AATATCTCTT CTGCAAAAGG CCTGGACGTT TGGGACAGCT GGCCATTACA 2401AAACGCTGAC GGCACTGTCG CAAACTATCA CGGCTACCAC ATCGTCTTTG 2451CATTAGCCGG AGATCCTAAA AATGCGGATG ACACATCGAT TTACATGTTC 2501TATCAAAAAG TCGGCGAAAC TTCTATTGAC AGCTGGAAAA ACGCTGGCCG 2551CGTCTTTAAA GACAGCGACA AATTCGATGC AAATGATTCT ATCCTAAAAG 2601ACCAAACACA AGAATGGTCA GGTTCAGCCA CATTTACATC TGACGGAAAA 2651ATCCGTTTAT TCTACACTGA TTTCTCCGGT AAACATTACG GCAAACAAAC 2701ACTGACAACT GCACAAGTTA ACGTATCAGC ATCAGACAGC TCTTTGAACA 2751TCAACGGTGT AGAGGATTAT AAATCAATCT TTGACGGTGA CGGAAAAACG 2801TATCAAAATG TACAGCAGTT CATCGATGAA GGCAACTACA GCTCAGGCGA 2851CAACCATACG CTGAGAGATC CTCACTACGT AGAAGATAAA GGCCACAAAT 2901ACTTAGTATT TGAAGCAAAC ACTGGAACTG AAGATGGCTA CCAAGGCGAA 2951GAATCTTTAT TTAACAAAGC ATACTATGGC AAAAGCACAT CATTCTTCCG 3001TCAAGAAAGT CAAAAACTTC TGCAAAGCGA TAAAAAACGC ACGGCTGAGT 3051TAGCAAACGG CGCTCTCGGT ATGATTGAGC TAAACGATGA TTACACACTG 3101AAAAAAGTGA TGAAACCGCT GATTGCATCT AACACAGTAA CAGATGAAAT 3151TGAACGCGCG AACGTCTTTA AAATGAACGG CAAATGGTAC CTGTTCACTG 3201ACTCCCGCGG ATCAAAAATG ACGATTGACG GCATTACGTC TAACGATATT 3251TACATGCTTG GTTATGTTTC TAATTCTTTA ACTGGCCCAT ACAAGCCGCT 3301GAACAAAACT GGCCTTGTGT TAAAAATGGA TCTTGATCCT AACGATGTAA 3351CCTTTACTTA CTCACACTTC GCTGTACCTC AAGCGAAAGG AAACAATGTC 3401GTGATTACAA GCTATATGAC AAACAGAGGA TTCTACGCAG ACAAACAATC 3451AACGTTTGCG CCAAGCTTCC TGCTGAACAT CAAAGGCAAG AAAACATCTG 3501TTGTCAAAGA CAGCATCCTT GAACAAGGAC AATTAACAGT TAACAAATAA 3551AAACGCAAAA GAAAATGCCG ATNNCCGGTT TATTGACTAC CGGAAGCAGT 3601GTGACCGTGT GCTTCTCAAA TGCCTCAGGC TGTCTATGTG TGACTGTTGA 3651GCTGTAACAA GTTGTCTCAG GTGTTCAATT TCATGTTCTA GTTGCTTTGT 3701TTTACTGGTT TCACCTGTTC TATTAGGTGT TACATGCTGT TCATCTGTTA 3751CATTGTCGAT CTGTTCATGG TGAACAGCTT TAAATGCACC AAAAACTCGT 3801AAAAGCTCTG ATGTATCTAT CTTTTTTACA CCGTTTTCAT CTGTGCATAT 3851GGACAGTTTT CCCTTTGATA TCTAACGGTG AACAGTTGTT CTACTTTTGT 3901TTGTTAGTCT TGATGCTTCA CTGATAGATA CAAGAGCCAT AAGAACCTCA 3951GATCCTTCCG TATTTAGCCA GTATGTTCTC TAGTGTGGTT CGTTGTTTTT 4001GCGTGAGCCA TGAGAACGAA CCATTGAGAT CATGCTTACT TTGCATGTCA 4051CTCAAAAATT TTGCCTCAAA ACTGGTGAGC TGAATTTTTG CAGTTAAAGC 4101ATCGTGTAGT GTTTTTCTTA GTCCGTTACG TAGGTAGGAA TCTGATGTAA 4151TGGTTGTTGG TATTTTGTCA CCATTCATTT TTATCTGGTT GTTCTCAAGT 4201TCGGTTACGA GATCCATTTG TCTATCTAGT TCAACTTGGA AAATCAACGT 4251ATCAGTCGGG CGGCCTCGCT TATCAACCAC CAATTTCATA TTGCTGTAAG 4301TGTTTAAATC TTTACTTATT GGTTTCAAAA CCCATTGGTT AAGCCTTTTA 4351AACTCATGGT AGTTATTTTC AAGCATTAAC ATGAACTTAA ATTCATCAAG 4401GCTAATCTCT ATATTTGCCT TGTGAGTTTT CTTTTGTGTT AGTTCTTTTA 4451ATAACCACTC ATAAATCCTC ATAGAGTATT TGTTTTCAAA AGACTTAACA 4501TGTTCCAGAT TATATTTTAT GAATTTTTTT AACTGGAAAA GATAAGGCAA 4551TATCTCTTCA CTAAAAACTA ATTCTAATTT TTCGCTTGAG AACTTGGCAT 4601AGTTTGTCCA CTGGAAAATC TCAAAGCCTT TAACCAAAGG ATTCCTGATT 4651TCCACAGTTC TCGTCATCAG CTCTCTGGTT GCTTTAGCTA ATACACCATA 4701AGCATTTTCC CTACTGATGT TCATCATCTG AGCGTATTGG TTATAAGTGA 4751ACGATACCGT CCGTTCTTTC CTTGTAGGGT TTTCAATCGT GGGGTTGAGT 4801AGTGCCACAC AGCATAAAAT TAGCTTGGTT TCATGCTCCG TTAAGTCATA 4851GCGACTAATC GCTAGTTCAT TTGCTTTGAA AACAACTAAT TCAGACATAC 4901ATCTCAATTG GTCTAGGTGA TTTTAATCAC TATACCAATT GAGATGGGCT 4951AGTCAATGAT AATTACTAGT CCTTTTCCTT TGAGTTGTGG GTATCTGTAA 5001ATTCTGCTAG ACCTTTGCTG GAAAACTTGT AAATTCTGCT AGACCCTCTG 5051TAAATTCCGC TAGACCTTTG TGTGTTTTTT TTGTTTATAT TCAAGTGGTT 5101ATAATTTATA GAATAAAGAA AGAATAAAAA AAGATAAAAA GAATAGATCC 5151CAGCCCTGTG TATAACTCAC TACTTTAGTC AGTTCCGCAG TATTACAAAA 5201GGATGTCGCA AACGCTGTTT GCTCCTCTAC AAAACAGACC TTAAAACCCT 5251AAAGGCTTAA GTAGCACCCT CGCAAGCTCG GGCAAATCGC TGAATATTCC 5301TTTTGTCTCC GACCATCAGG CACCTGAGTC GCTGTCTTTT TCGTGACATT 5351CAGTTCGCTG CGCTCACGGC TCTGGCAGTG AATGGGGGTA AATGGCACTA 5401CAGGCGCCTT TTATGGATTC ATGCAAGGAA ACTACCCATA ATACAAGAAA 5451AGCCCGTCAC GGGCTTCTCA GGGCGTTTTA TGGCGGGTCT GCTATGTGGT 5501GCTATCTGAC TTTTTGCTGT TCAGCAGTTC CTGCCCTCTG ATTTTCCAGT 5551CTGACCACTT CGGATTATCC CGTGACAGGT CATTCAGACT GGCTAATGCA 5601CCCAGTAAGG CAGCGGTATC ATCAACAGGC TTACCCGTCT TACTGTCnGG 5651ATCGACGCTC TCCCTTATGC GACTCCTGCA TPKOV(pKO5)16-Jan-19968673 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 acetyltransferase13-103813-1038 Cm-R; region1048,10891048SmaI cloning site(s)1057 1057,1088 NotI cloning site(s) 1064-2456nonePssR Leftflanking region2457-2579nonec22 \"universal\" KO-tag2580-3287nonePssR right flanking region4079-40851068-1074 BamHI cloning site4091-40971099-1105 SalI cloning site 4115-45711119-1575 f1 (+) filamentous origin4658-65642129-3550 Bacillus subtilis sacB gene7131-86394139-4215 origin (pSC101)7399-83494407-5357 RepA (temperature-sensitive) pkovLength: 8673May 9, 1999 04:58Type: NCheck: 2716..CCCTTTCGTCTTCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTGCTACGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGAAAGCAAATTCGACCCGGTCGTCGGTTCAGGGCAGGGTCGTTAAATAGCCGCTTATGTCTATTGCTGGTTTANTCGGTACCCGGGGATCGCGGCCGCCCCGAAACCAACTGATGCAGTATCCCGGGCGCTACAACATGATCTCAGTGATGTTATCGGTCAGGAACAAGGAAAGCGAGGACTGGAAATTACCGCTGCTGGCGGGCACAACCTTTTACTGATTGGGCCGCCGGGAACAGGTAAAACAATGCTCGCCAGCCGTATTAATGGCCTGTTGCCAGATTTAAGCAATGAAGAGGCACTGGAGAGTGCTGCGATATTAAGTCTGGTAAATGCTGAATCAGTACAAAAACAATGGCGGCAGCGCCCGTTCCGCTCACCTCATCACAGTGCATCGTTAACTGCGATGGTAGGCGGTGGCGCAATTCCAGGGCCCGGTGAAATTTCGCTGGCGCATAACGGCGTGCTTTTTCTTGATGAGCTACCTGAATTTGAACGGCGTACACTGGATGCCTTGCGAGAGCCGATTGAATCCGGGCAGATCCATCTTTCACGCACACGAGCAAAAATAACCTATCCAGCCCGTTTCCAGCTTGTTGCGGCGATGAATCCCAGCCCTACCGGACATTATCAGGGAAACCATAACCGCTGCACGCCAGAACAGACATTACGTTATCTCAACCGGCTCTCGGGGCCCTTTCTCGACCGCTTCGATCTCTCACTGGAGATCCCATTACCACCCCCCGGCATTTTGAGTAAAACGGTAGTGCCGGGAGAAAGCAGCGCCACCGTTAAACAACGCGTAATGGCCGCCAGAGAGCGCCAATTTAAGCGGCAGAATAAACTGAATGCCTGGCTGGATAGTCCGGAAATACGCCAATTCTGCAAACTTGAGAGCGAAGATGCGATGTGGTTGGAAGGAACACTGATCCATCTGGGGTTATCGATTCGTGCCTGGCAGCGGTTATTGAAAGTTGCACGAACCATTGCTGATATTGATCAGTCTGACATTATCACACGTCAGCATTTGCAGGAGGCAGTTAGCTATCGAGCGATTGACCGTTTGCTCATCCATCTGCAGAAACTACTGACATAAAAAAAGGGCATTTCGCCCTTTTTATTAATCGTCAGAATCGGTGTAGTCTTCAGCACCTTCAACCTGCGGTTTACCGCGGAAAGGGTGTGAAAACGTTTTGGACGCTTAATACGCGTCATATACTTGGACCACACGCGTTCTGCTTCTGTCACTGGCTCACGTTCGCCACGGCATACTGCTACGAAGAGTTTCTCTTCCTCGGTAACCGGCTCGCGTTTGCCAAGATCCAACTCATTGAAGGCATAACCATGACGCTCAAGCAGTTGTGCCTCTTTGATGGTGAAATCACCATGACGAGAGAATCCACGTGGATAATGTTTATTGTCGAAATATCGATTAGTCGTCGTAAAGCTTTCCGCCATCCTGCACGCTCCTAATTCTTTGACCGAGCTAGTTATGGCGCGGAGTATTAGTTACGCTTGACAGAGTGTAAAACAAAACATTTAAATCATAACGACAAATAATTTTGCGGAGAGCACTGTGGATGTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTGGTAAAACAGCTGCATGAACGCCAGCTTGATCTTCTTATTACCACTGAAGCGCCCAAAATGGACGAATTTAGTAGTCAGTTGCTGGGATATTTCACTTTAGCGCTTTATACCAGTGCCCCTTCAAAACTAAAGGGAGATCTTAATTATCTGCGACTTGAGTGGGGGCCAGATTTTCAACAGCATGAGGCAGGTTTGATCGGTGCTGACGAAGTGCCCATTCTGACAACCAGTTCTGCTGAACTGGCACAGCAACAGATTGCGATGCTTAATGGTTGCACCTGGCTACCCGTCAGCTGGGCGCGTAAAAAAGGCGGCCTGCATACCGTTGTCGATAGCACAACACTTTCACGGCCGCTTTATGCCATATGGCTGCAAAATAGCGATAAAAATGCGTTGATTCGCGATCTTTTGAAAATTAACGTGCTGGATGAAGTGTATTAATATGAATGGCTGGCAAGGATGCCGGTAGAAGGATTTACTTCGGAGAGGGTTATTTCAGATAAAAAAAATCCTTACGTTTCGCTAAGGATGATTTCTGGCAGGGGCGGAGAGACTCGAACTCCCAACACCCGGTTTTGGAGACCGGTGCTCTACCAATTGAACTACGCCCCTAATTAGGGTGGCGGAACGGACGGGACTCGAACCCGCGACCCCCTGCGTGACAGGCAGGTATTCTAACCGACTGAACTACCGCTCCACCGAATTCTTTTACAACCACCGGTTTTATGACCGGCTTACTGCTTAATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACGGCCGCCAGGCAAATTCTGTTTCATCAGACCGCTTCTGCGTTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATCTTCGGCGTTGTAAGGTTAAGCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTGCGCTTACACACCCGGCCTATCAACGTCGTCGTCTTCAACGTTCCTTCAGGACTCTCAAGGAGTCAGGGAGAACTCATCTCGGGGCAAGTTTCGTGCTTAGATGCTTTCAGCACTTATCTCTTCCGCATTTAGCTACCGGGCAGTGCCATTGGCATGACAACCCGAACACCAGTGATGCGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCCCCTCAGTTCTCCAGCGCCCACGGCAGATAGGGACCGAACTGTCTCACGACGTTCTAAACCCAGCTCGCGTACCACTTTAAATGGCGAACAGCCATACCCTTGGGACCTACTTCAGCTCCAGGATGTGATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACTCTTGGGCGGTATCAGCCTGTTATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAACCACCGGATCACTATGACCTGCTTTCGCACCTGGGATCCTCTAGAGTCGACCGGNGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATNNCANGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTCCTGAATTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCAAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATNNCCGGTTTATTGACTACCGGAAGCAGTGTGACCGTGTGCTTCTCAAATGCCTCAGGCTGTCTATGTGTGACTGTTGAGCTGTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGTTTTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACATTGTCGATCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGTAAAAGCTCTGATGTATCTATCTTTTTTACACCGTTTTCATCTGTGCATATGGACAGTTTTCCCTTTGATATCTAACGGTGAACAGTTGTTCTACTTTTGTTTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCTCAGATCCTTCCGTATTTAGCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGCTTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAAAGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTGGTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCCTTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATCTCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCTCATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTAACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAACTTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCACAGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGATGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGGTTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGTTAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATCTCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTACTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACTTGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATATTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCCAGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAAGCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCTTTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACTACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACGGGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCAGCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACAGGCTTACCCGTCTTACTGTCNGGATCGACGCTCTCCCTTATGCGACTCCTGCATThese files have been constructed from various GenBank files, are not primary data, and have not been confirmed.
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