pKD46

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  • Model: PVT6005
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pKD46

PVT6005   2ug                                    Lot no 1901093                            Data     CoA

Store at -20                                    

 

pKD46 Information

Promoter: araBAD promoter

Replicon: pSC101 ori

Terminator: lambda tL3 Terminator

Prokaryotic resistance: ampicillin Amp

Cloned strain: Escherichia coli DH5 alpha

Culture conditions: 30 degrees centigrade

Expression host: Escherichia coli

Inducement: Arabia sugar

Use:Homologous recombination HR plasmid

Use:Lactic Acid Bacteria Plasmids

 

pKD46 Description

PKD46 plasmid is the most widely used Red homologous recombination plasmid currently. The plasmid contains exo, bet and gam genes regulated by Arabia sugar promoter, and its replicon is low copy temperature sensitive replicon, which is convenient for plasmid elimination.

Related 3 Enzymesto the knockout of homologous recombination genes:

1.Exo protein

Exo protein is an exonuclease, single subunit molecular weight is 24 kD, the active form of Exo protein is a cyclic trimer, a hollow passage, one end of the channel can accommodate a double stranded DNA molecule, the other end can only contain a single stranded DNA. The Exo protein can be combined at the end of the double stranded DNA and degrade DNA from the 5 'end of the DNA double strand to the 3' end, causing the DNA molecule to form a 3 'sticky end.

2. Beta protein

Beta protein plays a decisive role in Red homologous recombination, is mediated by complementary single strand annealing of the DNA function, which is an annealing protein, the molecular weight of single subunit was 25.8 kD. In solution, Beta protein spontaneously forms ring structure, tightly bound to the 3 'prominent end of single stranded DNA, preventing DNA from being degraded by single chain nuclease and mediating the annealing of complementary single strand DNA. After the double stranded DNA was annealed, the Beta protein was dissociated from the double strand of DNA.

3. Gam protein

Gam protein is a polypeptide molecule of 16kD, can be combined with RecBCD exonuclease, inhibiting the degradation of exogenous DNA, to prevent foreign DNA into cells after degradation by the host.
 

pKD46  Reference

[1] Datsenko KA1, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000,97(12):6640-6645.
[2] Doublet B, Douard G, Targant H, et al. Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains. J Microbiol Methods. 2008,75(2):359-361. Doi: 10.1016/j.mimet.2008.06.010

 

pKD46 Sequences

LOCUS Exported 6329 bp ds-DNA circular SYN 18-09-2015

DEFINITION synthetic circular DNA

ACCESSION .

VERSION .

KEYWORDS pKD46

SOURCE synthetic DNA construct

ORGANISM synthetic DNA construct

REFERENCE 1 (bases 1 to 6329)

AUTHORS .

TITLE Direct Submission

JOURNAL Exported 2015-9-18 from SnapGene Viewer 2.8.1

 

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)"

terminator 3129..3373

/note="lambda tL3 terminator"

/note="transcription terminator tL3 from phage lambda"

CDS complement(3496..4446)

/codon_start=1

/gene="rep101"

/product="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

//

 

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  1. This product is FOR RESEARCH USE ONLY!

 

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