基因敲出質(zhì)粒圖譜

1、（1）PKD46載體基本信息出品公司:-載體名稱:pKD46質(zhì)粒類型:細菌表達；基因敲除系統(tǒng)高拷貝/低拷貝:-啟動子:-克隆方法:多克隆位點，限制性內(nèi)切酶載體大小:6329 bp5' 測序引物及序列:-3' 測序引物及序列:-載體標簽:-載體抗性:-篩選標記:-備注:pKD46 is more efficient than pKD20 for making gene disruptions.產(chǎn)品目錄號:-穩(wěn)定性:-組成型:-病毒/非病毒:-LOCUS pKD46 6329 bp DNA circular SYN 11-SEP-2001DEFINITION Red recombi

2、nase plasmid pKD46, complete sequence.ACCESSION AY048746VERSION AY048746.1 GI:15554345KEYWORDS .SOURCE Red recombinase plasmid pKD46 ORGANISM Red recombinase plasmid pKD46 other sequences; artificial sequences; vectors.REFERENCE 1 (bases 1 to 6329) AUTHORS Datsenko,K.A. and Wanner,B.L. TITLE One-ste

3、p inactivation of chromosomal genes in Escherichia coli K-12 using PCR products JOURNAL Proc. Natl. Acad. Sci. U.S.A. 97 (12), 6640-6645 (2000) PUBMED 10829079REFERENCE 2 (bases 1 to 6329) AUTHORS Datsenko,K.A. and Wanner,B.L. TITLE Direct Submission JOURNAL Submitted (29-JUL-2001) Biological Scienc

4、es, Purdue University, Lilly Hall of Life Sciences, West Lafayette, IN 47907, USAFEATURES Location/Qualifiers source 1.6329 /organism="Red recombinase plasmid pKD46" /mol_type="genomic DNA" /db_xref="taxon:170495" CDS complement(11.889) /codon_start=1 /transl_table=11 /

5、product="araC protein" /protein_id="AAL02045.1" /db_xref="GI:15554346" misc_feature complement(1040.1068) /note="Pc" misc_feature 1165.1192 /note="ParaB" CDS 1244.1660 /note="recombination;138" /codon_start=1 /transl_table=11 /product="

6、;gam protein" /protein_id="AAL02046.1" /db_xref="GI:15554347" CDS 1666.2451 /note="recombination;261" /codon_start=1 /transl_table=11 /product="bet protein" /protein_id="AAL02047.1" /db_xref="GI:15554348" CDS 2448.3128 /note="exo&

7、quot; /codon_start=1 /transl_table=11 /product="exonuclease" /protein_id="AAL02048.1" /db_xref="GI:15554349" misc_feature 3118.3387 /note="tL3" CDS 3125.3307 /note="60a" /codon_start=1 /transl_table=11 /product="orf60a protein" /protein_id=

8、"AAL02051.1" /db_xref="GI:15554352" CDS complement(3496.4446) /codon_start=1 /transl_table=11 /product="repA101 protein" /protein_id="AAL02049.1" /db_xref="GI:15554350" rep_origin 4450.5190 /note="oriR101" CDS complement(5338.6198) /note=&q

9、uot;bla; ampicillin resistance" /codon_start=1 /transl_table=11 /product="beta-lactamase" /protein_id="AAL02050.1" /db_xref="GI:15554351"ORIGIN 1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcga

10、gaaa tagagttgat 121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca 181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 361

11、tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg 541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 601 aaacccactg gtgatacca

12、t tcgcgagcct ccggatgacg accgtagtga tgaatctctc 661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca 721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 841 cattaaacga gtatcccggc agcaggggat cattttg

13、cgc ttcagccata cttttcatac 901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg 961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt 1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca 1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gca

14、tttttat 1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga 1261 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc 1321 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat 1381 tca

15、ggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga 1441 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc 1501 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt 1561 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac 1621 cattgctcac caccaggt

16、tg atattgattc agaggtataa aacgaatgag tactgcactc 1681 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa 1741 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc 1801 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc 1861 tttcctgata agcagaatgg catcgttccg gt

17、ggtgggcg ttgatggctg gtcccgcatc 1921 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca 1981 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa 2041 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg 2101 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccg

18、tct ggccttcgga 2161 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact 2221 gcagaacgtc agccggaacg cgacatcact ccggttaacg atgaaaccat gcaggagatt 2281 aacactctgc tgatcgccct ggataaaaca tgggatgacg acttattgcc gctctgttcc 2341 cagatatttc gccgcgacat tcgtgcatcg tcagaactga cacaggccga agcagtaaaa 2

19、401 gctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca 2461 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc 2521 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacccc 2581 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg 2641 tttgcaccgg t

20、gtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg 2701 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga 2761 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg 2821 gcaacggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg 2881 gtggtttcga ggccataaag tcagct

21、taca tggcccaggt gcagtacagc atgtgggtga 2941 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc 3001 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg 3061 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat 3121 ggcgatgacg catcctcacg ataatatccg ggtaggcgca

22、atcactttcg tctactccgt 3181 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca 3241 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt 3301 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg 3361 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttg

23、atatg 3421 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca 3481 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 3541 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 3601 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 3661 ttttc

24、ttagt ccgttacgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 3721 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 3781 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt 3841 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 3901 ctcatggtag ttattttcaa

25、 gcattaacat gaacttaaat tcatcaaggc taatctctat 3961 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 4021 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa 4081 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa 4141 cttggcatag tttgtccact ggaaaatctc aaag

26、ccttta accaaaggat tcctgatttc 4201 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct 4261 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct 4321 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 4381 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaa

27、a caactaattc 4441 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 4501 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 4561 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg 4621 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 468

28、1 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 4741 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 4801 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata 4861 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc 4921 gctgcgctca cgg

29、ctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 4981 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 5041 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 5101 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 5161 tgcacccagt aaggcagcgg tatcatca

30、ac ggggtctgac gctcagtgga acgaaaactc 5221 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5281 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5341 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5401 tgcctgactc cccgtcgtgt agataactac gatacgggag gg

31、cttaccat ctggccccag 5461 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5521 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5581 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5641 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcatt

32、cag 5701 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5761 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5821 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5881 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5941 ttgcccg

33、gcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6001 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6061 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6121 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6181 gaaatgttga atactcatac t

34、cttcctttt tcaatattat tgaagcattt atcagggtta 6241 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6301 gcgcacattt ccccgaaaag tgccacctg摘要：目前應(yīng)用最廣泛的Red同源重組系統(tǒng)質(zhì)粒是pKD46，該質(zhì)粒含有受阿拉伯糖啟動子調(diào)控的exo、bet、gam基因，具有氨芐青霉素抗性，其復(fù)制子為低拷貝溫敏型復(fù)制子，便于質(zhì)粒的消除。pKD46質(zhì)粒序列全長6329 bp，詳細序列見GenBank進入號：AY048746。關(guān)鍵詞： Red&

35、#160;, 同源重組 , 基因敲除 , 質(zhì)粒圖譜pKD46質(zhì)粒是目前應(yīng)用最廣泛的Red同源重組質(zhì)粒，該質(zhì)粒含有受阿拉伯糖啟動子調(diào)控的exo、bet、gam基因，具有氨芐青霉素抗性，其復(fù)制子為低拷貝溫敏型復(fù)制子，便于質(zhì)粒的消除。pKD46質(zhì)粒序列全長6329 bp，詳細序列見GenBank進入號：AY048746。質(zhì)粒類型：Red同源重組基因敲除載體克隆方法：多克隆位點，限制性內(nèi)切酶拷貝數(shù)：低拷貝載體大?。?329 bp（GenBank進入號：AY048746）載體抗性：Ampicillin (氨芐青霉素)備注：30°C培養(yǎng)與同源重組

36、基因敲除相關(guān)的3個酶：1、Exo蛋白Exo蛋白是一種核酸外切酶，單亞基的分子量為24 kD，Exo蛋白的活性形式是一種環(huán)狀三聚物分子，中間有一中空的通道，通道的一端可容納雙鏈DNA分子，另一端只可容納單鏈DNA。Exo蛋白可結(jié)合在雙鏈DNA的末端，從DNA雙鏈的5端向3端降解DNA，使DNA分子形成3粘性末端。2、Beta蛋白Beta蛋白在Red同源重組中起著決定性作用，具有介導(dǎo)互補單鏈DNA退火的功能，是一種退火蛋白，單亞基的分子量為25.8 kD。在溶液中，Beta蛋白自發(fā)地形成環(huán)狀結(jié)構(gòu)，緊緊地結(jié)合在單鏈DNA的3突出端，防止DNA被單鏈核酸酶降解，并介導(dǎo)互補單鏈DNA的退火。雙鏈DNA退火完成后，Beta蛋白從DNA雙鏈上解離下來。3、Gam蛋白Gam蛋白為16kD的多肽分子，可與RecBCD核酸外切酶結(jié)合，抑制其對外源DNA的降解作用，防止外源DNA 進入細胞后被宿主降解。參考文獻：1 Datsenko KA1, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coliK-12 using PCR products. Proc Natl Acad Sci USA. 2000,97(12):6640-6645.2 Doublet