| 质粒名称: | pSNK |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 135193 |
| 存储实验室: | Hongqing Fang |
|
载体骨架基本信息 |
|
| 载体名称: | pKOBEG |
| 完整质粒大小: | 7454 bp |
| 载体类型: | Bacterial Expression, Synthetic Biology |
| 载体抗性: | Kanamycin |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | I-SceI lamba Red |
| 物种来源: | Synthetic |
| 基因突变: | 无 |
| 启动子: | araBAD |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NheI(未破坏) |
| 3’克隆位点: | HindIII(未破坏) |
| 5’测序引物 | 未知 |
|
文献引用方法 |
|
| 材料和方法部分: | pSNK was a gift from Hongqing Fang (Addgene plasmid # 135193 ; http://n2t.net/addgene:135193 ; RRID:Addgene_135193) |
| 参考文献部分: | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome. Wei Y, Deng P, Mohsin A, Yang Y, Zhou H, Guo M, Fang H. PLoS One. 2017 Oct 24;12(10):e0186891. doi: 10.1371/journal.pone.0186891. eCollection 2017. 10.1371/journal.pone.0186891 PubMed 29065183 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pSNK 7454 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Arabinose inducible expression of lamba-Red recombinase proteins and
I-SceI. Contains kamamycin resistance cassette.
ACCESSION .
VERSION .
KEYWORDS pSNK
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7454)
AUTHORS Wei Y, Deng P, Mohsin A, Yang Y, Zhou H, Guo M, Fang H
TITLE An electroporation-free method based on Red recombineering for
markerless deletion and genomic replacement in the Escherichia coli
DH1 genome.
JOURNAL PLoS One. 2017 Oct 24;12(10):e0186891. doi:
10.1371/journal.pone.0186891. eCollection 2017.
PUBMED 29065183
REFERENCE 2 (bases 1 to 7454)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7454
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 430..846
/codon_start=1
/gene="gam"
/product="inhibitor of the host RecBCD nuclease in the
lambda Red system"
/label=Gam
/note="protects the double-stranded DNA substrate for
recombination"
/translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
CDS 852..1637
/codon_start=1
/gene="bet"
/product="single-stranded DNA binding recombinase in the
lambda Red system"
/label=Beta
/note="mediates pairing of a single-stranded DNA with a
complementary target"
/translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
CDS 1634..2314
/codon_start=1
/gene="exo"
/product="5' to 3' double-stranded DNA exonuclease in the
lambda Red system"
/label=Exo
/note="creates 3' single-stranded DNA tails"
/translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR
SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
EQWR"
CDS complement(2812..3762)
/codon_start=1
/gene="rep101 encoding an A56V mutation"
/product="temperature-sensitive version of the RepA protein
needed for replication with the pSC101 origin (Armstrong et
al., 1984)"
/label=Rep101(Ts)
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin 3810..4032
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
primer_bind complement(4815..4834)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 4826..5641
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind complement(4899..4918)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
CDS complement(5906..6784)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 6811..7095
/gene="araBAD"
/label=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)"
primer_bind 7018..7037
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
ORIGIN
1 aagcattcaa caaactggct aacctgttca tcgttaacaa caaaaaaacc atcccgaaca
61 acctggttga aaactacctg accccgatgt ctctggcata ctggttcatg gatgatggtg
121 gtaaatggga ttacaacaaa aactctacca acaaatcgat cgtactgaac acccagtctt
181 tcactttcga agaagtagaa tacctggtta agggtctgcg taacaaattc caactgaact
241 gttacgtaaa aatcaacaaa aacaaaccga tcatctacat cgattctatg tcttacctga
301 tcttctacaa cctgatcaaa ccgtacctga tcccgcagat gatgtacaaa ctgccgaaca
361 ctatctcctc cgaaactttc ctgaaataag ctagaagatg tttcgtgaag ccgtcgacgc
421 ttataaaaaa tggatattaa tactgaaact gagatcaagc aaaagcattc actaaccccc
481 tttcctgttt tcctaatcag cccggcattt cgcgggcgat attttcacag ctatttcagg
541 agttcagcca tgaacgctta ttacattcag gatcgtcttg aggctcagag ctgggcgcgt
601 cactaccagc agctcgcccg tgaagagaaa gaggcagaac tggcagacga catggaaaaa
661 ggcctgcccc agcacctgtt tgaatcgcta tgcatcgatc atttgcaacg ccacggggcc
721 agcaaaaaat ccattacccg tgcgtttgat gacgatgttg agtttcagga gcgcatggca
781 gaacacatcc ggtacatggt tgaaaccatt gctcaccacc aggttgatat tgattcagag
841 gtataaaacg aatgagtact gcactcgcaa cgctggctgg gaagctggct gaacgtgtcg
901 gcatggattc tgtcgaccca caggaactga tcaccactct tcgccagacg gcatttaaag
961 gtgatgccag cgatgcgcag ttcatcgcat tactgatcgt tgccaaccag tacggcctta
1021 atccgtggac gaaagaaatt tacgcctttc ctgataagca gaatggcatc gttccggtgg
1081 tgggcgttga tggctggtcc cgcatcatca atgaaaacca gcagtttgat ggcatggact
1141 ttgagcagga caatgaatcc tgtacatgcc ggatttaccg caaggaccgt aatcatccga
1201 tctgcgttac cgaatggatg gatgaatgcc gccgcgaacc attcaaaact cgcgaaggca
1261 gagaaatcac ggggccgtgg cagtcgcatc ccaaacggat gttacgtcat aaagccatga
1321 ttcagtgtgc ccgtctggcc ttcggatttg ctggtatcta tgacaaggat gaagccgagc
1381 gcattgtcga aaatactgca tacactgcag aacgtcagcc ggaacgcgac atcactccgg
1441 ttaacgatga aaccatgcag gagattaaca ctctgctgat cgccctggat aaaacatggg
1501 atgacgactt attgccgctc tgttcccaga tatttcgccg cgacattcgt gcatcgtcag
1561 aactgacaca ggccgaagca gtaaaagctc ttggattcct gaaacagaaa gccgcagagc
1621 agaaggtggc agcatgacac cggacattat cctgcagcgt accgggatcg atgtgagagc
1681 tgtcgaacag ggggaagatg cgtggcacaa attacggctc ggcgtcatca ccgcttcaga
1741 agttcacaac gtgatagcaa aaccccgctc cggaaagaag tggcctgaca tgaaaatgtc
1801 ctacttccac accctgcttg ctgaggtttg caccggtgtg gctccggaag ttaacgctaa
1861 agcactggcc tggggaaaac agtacgagaa cgacgccaga accctgtttg aattcacttc
1921 cggcgtgaat gttactgaat ccccgatcat ctatcgcgac gaaagtatgc gtaccgcctg
1981 ctctcccgat ggtttatgca gtgacggcaa cggccttgaa ctgaaatgcc cgtttacctc
2041 ccgggatttc atgaagttcc ggctcggtgg tttcgaggcc ataaagtcag cttacatggc
2101 ccaggtgcag tacagcatgt gggtgacgcg aaaaaatgcc tggtactttg ccaactatga
2161 cccgcgtatg aagcgtgaag gcctgcatta tgtcgtgatt gagcgggatg aaaagtacat
2221 ggcgagtttt gacgagatcg tgccggagtt catcgaaaaa atggacgagg cactggctga
2281 aattggtttt gtatttgggg agcaatggcg atgacgcatc ctcacgataa taagcttcct
2341 gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac agcatccttg aacaaggaca
2401 attaacagtt aacaaataaa aacgcaaaag aaaatgccga tattgactac cggaagcagt
2461 gtgaccgtgt gcttctcaaa tgcctgattc aggctgtcta tgtgtgactg ttgagctgta
2521 acaagttgtc tcaggtgttc aatttcatgt tctagttgct ttgttttact ggtttcacct
2581 gttctattag gtgttacatg ctgttcatct gttacattgt cgatctgttc atggtgaaca
2641 gctttaaatg caccaaaaac tcgtaaaagc tctgatgtat ctatcttttt tacaccgttt
2701 tcatctgtgc atatggacag ttttcccttt gatatgtaac ggtgaacagt tgttctactt
2761 ttgtttgtta gtcttgatgc ttcactgata gatacaagag ccataagaac ctcagatcct
2821 tccgtattta gccagtatgt tctctagtgt ggttcgttgt ttttgcgtga gccatgagaa
2881 cgaaccattg agatcatact tactttgcat gtcactcaaa aattttgcct caaaactggt
2941 gagctgaatt tttgcagtta aagcatcgtg tagtgttttt cttagtccgt tatgtaggta
3001 ggaatctgat gtaatggttg ttggtatttt gtcaccattc atttttatct ggttgttctc
3061 aagttcggtt acgagatcca tttgtctatc tagttcaact tggaaaatca acgtatcagt
3121 cgggcggcct cgcttatcaa ccaccaattt catattgctg taagtgttta aatctttact
3181 tattggtttc aaaacccatt ggttaagcct tttaaactca tggtagttat tttcaagcat
3241 taacatgaac ttaaattcat caaggctaat ctctatattt gccttgtgag ttttcttttg
3301 tgttagttct tttaataacc actcataaat cctcatagag tatttgtttt caaaagactt
3361 aacatgttcc agattatatt ttatgaattt ttttaactgg aaaagataag gcaatatctc
3421 ttcactaaaa actaattcta atttttcgct tgagaacttg gcatagtttg tccactggaa
3481 aatctcaaag cctttaacca aaggattcct gatttccaca gttctcgtca tcagctctct
3541 ggttgcttta gctaatacac cataagcatt ttccctactg atgttcatca tctgaacgta
3601 ttggttataa gtgaacgata ccgtccgttc tttccttgta gggttttcaa tcgtggggtt
3661 gagtagtgcc acacagcata aaattagctt ggtttcatgc tccgttaagt catagcgact
3721 aatcgctagt tcatttgctt tgaaaacaac taattcagac atacatctca attggtctag
3781 gtgattttaa tcactatacc aattgagatg ggctagtcaa tgataattac tagtcctttt
3841 cctttgagtt gtgggtatct gtaaattctg ctagaccttt gctggaaaac ttgtaaattc
3901 tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt ttttttgttt atattcaagt
3961 ggttataatt tatagaataa agaaagaata aaaaaagata aaaagaatag atcccagccc
4021 tgtgtataac tcactacttt agtcagttcc gcagtattac aaaaggatgt cgcaaacgct
4081 gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc ttaagtagca ccctcgcaag
4141 ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat caggcacctg agtcgctgtc
4201 tttttcgtga cattcagttc gctgcgctca cggctctggc agtgaatggg ggtaaatggc
4261 actacaggcg ccttttatgg attcatgcaa ggaaactacc cataatacaa gaaaagcccg
4321 tcacgggctt ctcagggcgt tttatggcgg gtctgctatg tggtgctatc tgactttttg
4381 ctgttcagca gttcctgccc tctgattttc cagtctgacc acttcggatt atcccgtgac
4441 aggtcattca gactggctaa tgcacccagt aaggcagcgg tatcatcaac aggcttaccc
4501 gtcttactgt cggggatcga cgctctccct tatgcgactc ctgcaccttt cgtcttcgaa
4561 taaatacctg tgacggaaga tcacttcgca gataaaataa atcctggagt tggtagctct
4621 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
4681 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
4741 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta
4801 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag
4861 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
4921 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
4981 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
5041 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
5101 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga
5161 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
5221 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
5281 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
5341 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
5401 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
5461 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
5521 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
5581 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
5641 agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
5701 ttccgcgcac atttccccga aaagtgccac ctgtcgtcgg ttcagggcag ggtcgttaaa
5761 tagccgctta tgtctattgc tggtctcggt acccggggat cctctagagt cgacctgcat
5821 aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc
5881 cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt
5941 cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg
6001 agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg
6061 tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc
6121 taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct
6181 gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag
6241 cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc
6301 gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt
6361 gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg
6421 ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg
6481 cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt
6541 agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc
6601 gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca
6661 ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac
6721 ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag
6781 ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac
6841 attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta
6901 ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg
6961 tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg
7021 ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct
7081 ctactgtttc tccatacccg tttttttggg ctagcaggag ggtacctata tgcatatgaa
7141 aatcatcaaa aaaaaccagg taatgaacct cggtccgaac tctaaactgc tgaaagaata
7201 caaatcccag ctgatcgaac tgaacatcga acagttcgaa gcaggtatcg gtctgatcct
7261 gggtgatgct tacatccgtt ctcgtgatga aggtaaaacc tactgtatgc agttcgagtg
7321 gaaaaacaaa gcatacatgg accacgtatg tctgctgtac gatcagtggg tactgtcccc
7381 gccgcacaaa aaagaacgtg ttaaccacct gggtaacctg gtaatcacct ggggcgccca
7441 gactttcaaa cacc
//
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