應用pKO3載體進行基因置換和基因敲除重組 價　　格：￥5920 貨　　號：Biovector-pKO3 產　　地：北京 點擊詢問我要采購 竭誠為您服務！ 普如汀生物技術有限公司 聯系人：Dr.Xu, Biovector NTCC Inc. 點擊這里給我發消息 電話：010-53513060, 18901268599 郵件：Biovector@163.com 手機：18901268599 地址：北京 已注冊 詳細信息 咨詢記錄(共0條) 分享 新浪微博 騰訊微博 QQ空間 豆瓣 人人網 丁香客 微信 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 pKO3 vector map. Biovector NTCC Inc. 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 pKO3_CLONING_SITE, Biovector NTCC Inc. 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.