| 质粒名称: | pK18msB |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 177839 |
| 存储实验室: | Christopher Johnson |
|
载体骨架基本信息 |
|
| 载体名称: | pK18sB |
| 空载体大小: | 3282 bp |
| 完整质粒大小: | 3282 bp |
| 载体类型: | Bacterial gene replacement |
| 筛选标记: | Counterselection: Sucrose |
| 载体抗性: | Kanamycin, 50 μg/mL |
| 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: | 未知 |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | Special M13R binding site, M13R: CAGGAAACAGCTATGAC |
| 3’测序引物 | M13F: TGTAAAACGACGGCCAGT |
|
文献引用方法 |
|
| 材料和方法部分: | pK18msB was a gift from Christopher Johnson (Addgene plasmid # 177839 ; http://n2t.net/addgene:177839 ; RRID:Addgene_177839) |
| 参考文献部分: | Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering. Ling C, Peabody GL, Salvachua D, Kim YM, Kneucker CM, Calvey CH, Monninger MA, Munoz NM, Poirier BC, Ramirez KJ, St John PC, Woodworth SP, Magnuson JK, Burnum-Johnson KE, Guss AM, Johnson CW, Beckham GT. Nat Commun. 2022 Aug 22;13(1):4925. doi: 10.1038/s41467-022-32296-y. 10.1038/s41467-022-32296-y PubMed 35995792 |
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 pK18msB 3282 bp ds-DNA circular SYN 03-DEC-2021
DEFINITION Plasmid for gene replacement using kanamycin/sucrose
selection/counterselection. Derived from pK18mobsacB but reduced in
size..
ACCESSION .
VERSION .
KEYWORDS pK18msB
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3282)
TITLE Improving formate conversion by Cupriavidus necator
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 3282)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Dec 3, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..3282
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 292..311
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
CDS 429..1850
/codon_start=1
/gene="Bacillus subtilis sacB"
/product="secreted levansucrase that renders bacterial
growth sensitive to sucrose"
/label=SacB
/note="negative selection marker"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
oriT 1867..1975
/note="incP origin of transfer"
rep_origin 2127..2715
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2616..2635
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2727..2743
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2727..2743
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
misc_feature 2752..2808
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(2812..2829)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2812..2828)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS 2910..422
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(2964..2983)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
ORIGIN
1 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
61 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
121 tcgcgccagc cgaactgttc gccaggctca aggcgcggat gcccgacggc gaggatctcg
181 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
241 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
301 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
361 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
421 gagcgacgat gaacatcaaa aagtttgcaa aacaagcaac agtattaacc tttactaccg
481 cactgctggc aggaggcgca actcaagcgt ttgcgaaaga aacgaaccaa aagccatata
541 aggaaacata cggcatttcc catattacac gccatgatat gctgcaaatc cctgaacagc
601 aaaaaaatga aaaatatcaa gtttctgaat ttgattcgtc cacaattaaa aatatctctt
661 ctgcaaaagg cctggacgtt tgggacagct ggccattaca aaacgctgac ggcactgtcg
721 caaactatca cggctaccac atcgtctttg cattagccgg agatcctaaa aatgcggatg
781 acacatcgat ttacatgttc tatcaaaaag tcggcgaaac ttctattgac agctggaaaa
841 acgctggccg cgtctttaaa gacagcgaca aattcgatgc aaatgattct atcctaaaag
901 accaaacaca agaatggtca ggttcagcca catttacatc tgacggaaaa atccgtttat
961 tctacactga tttctccggt aaacattacg gcaaacaaac actgacaact gcacaagtta
1021 acgtatcagc atcagacagc tctttgaaca tcaacggtgt agaggattat aaatcaatct
1081 ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt catcgatgaa ggcaactaca
1141 gctcaggcga caaccatacg ctgagagatc ctcactacgt agaagataaa ggccacaaat
1201 acttagtatt tgaagcaaac actggaactg aagatggcta ccaaggcgaa gaatctttat
1261 ttaacaaagc atactatggc aaaagcacat cattcttccg tcaagaaagt caaaaacttc
1321 tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg cgctctcggt atgattgagc
1381 taaacgatga ttacacactg aaaaaagtga tgaaaccgct gattgcatct aacacagtaa
1441 cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac ctgttcactg
1501 actcccgcgg atcaaaaatg acgattgacg gcattacgtc taacgatatt tacatgcttg
1561 gttatgtttc taattcttta actggcccat acaagccgct gaacaaaact ggccttgtgt
1621 taaaaatgga tcttgatcct aacgatgtaa cctttactta ctcacacttc gctgtacctc
1681 aagcgaaagg aaacaatgtc gtgattacaa gctatatgac aaacagagga ttctacgcag
1741 acaaacaatc aacgtttgcg ccgagcttcc tgctgaacat caaaggcaag aaaacatctg
1801 ttgtcaaaga cagcatcctt gaacaaggac aattaacagt taacaaataa tcagaccccg
1861 tagaaacggc cagcctcgca gagcaggatt cccgttgagc accgccaggt gcgaataagg
1921 gacagtgaag aaggaacacc cgctcgcggg tgggcctact tcacctatcc tgcccggctg
1981 acgccgttgg atacaccaag gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg
2041 tgcgaaaaag gatggatata ccgaaaaaat cgctataatg accccgaagc agggttatgc
2101 agcggaaaag agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
2161 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
2221 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
2281 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
2341 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
2401 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
2461 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
2521 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
2581 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
2641 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
2701 cggagcctat ggaaaaacgc ctcacacagg aaacagctat gacatgatta cgaattcgag
2761 ctcggtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cactggccgt
2821 cgttttacaa cgtcgtgacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
2881 ccctgcaaac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
2941 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
3001 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
3061 ccgacctgtc cggtgccctg aatgaactcc aagacgaggc agcgcggcta tcgtggctgg
3121 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
3181 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
3241 agaaagtatc catcatggct gatgcaatgc ggcggctgca ta
//
