pKD46大肠敲除质粒

基本信息

启动子:	araBAD promoter
复制子:	pSC101 ori
终止子:	λ tL3 terminator
原核抗性:	氨苄青霉素Amp
克隆菌株:	大肠杆菌DH5α
培养条件:	LB培养基，30℃
表达宿主:	大肠杆菌
诱导方式:	阿拉伯糖

质粒简介

        pKD46质粒是目前应用最广泛的Red同源重组质粒，该质粒含有受阿拉伯糖启动子调控的exo、bet、gam基因，其复制子为低拷贝温敏型复制子，便于质粒的消除。
与同源重组基因敲除相关的3个酶：
        1、Exo蛋白：Exo蛋白是一种核酸外切酶，单亚基的分子量为24 kD，Exo蛋白的活性形式是一种环状三聚物分子，中间有一中空的通道，通道的一端可容纳双链DNA分子，另一端只可容纳单链DNA。Exo蛋白可结合在双链DNA的末端，从DNA双链的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同源重组中起着决定性作用，具有介导互补单链DNA退火的功能，是一种退火蛋白，单亚基的分子量为25.8 kD。在溶液中，Beta蛋白自发地形成环状结构，紧紧地结合在单链DNA的3’突出端，防止DNA被单链核酸酶降解，并介导互补单链DNA的退火。双链DNA退火完成后，Beta蛋白从DNA双链上解离下来。
        3、Gam蛋白：Gam蛋白为16kD的多肽分子，可与RecBCD核酸外切酶结合，抑制其对外源DNA的降解作用，防止外源DNA 进入细胞后被宿主降解。

质粒图谱

质粒序列

LOCUS       Exported                6329 bp ds-DNA     circular SYN 24-JUL-2017
DEFINITION  synthetic circular DNA
FEATURES             Location/Qualifiers
     source          1..6329
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(11..889)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /note="araC"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        916..1200
                     /gene="araBAD"
                     /note="araBAD promoter"
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     CDS             1244..1660
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /note="Gam"
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     CDS             1666..2451
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /note="Beta"
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             2448..3128
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /note="Exo"
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     terminator      3129..3373
                     /note="lambda tL3 terminator"
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(3496..4446)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /note="Rep101"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4494..4716
                     /note="pSC101 ori"
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(5338..6198)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6199..6303)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
       61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa 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 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 gtgataccat 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 cattttgcgc 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 gcatttttat
     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 tcaggatcgt 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 caccaggttg 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 gtggtgggcg 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 gtgcccgtct 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
     2401 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 tgtggctccg 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 tcagcttaca 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 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 ctttgatatg
     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 ttttcttagt 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 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 aaagccttta 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 gctttgaaaa 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
     4681 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 cggctctggc 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 tatcatcaac 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 ggcttaccat 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 cttcattcag
     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 ttgcccggcg 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 tcttcctttt tcaatattat tgaagcattt atcagggtta
     6241 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6301 gcgcacattt ccccgaaaag tgccacctg