| 质粒名称: | pKOV |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 25769 |
| 存储实验室: | George Church |
|
载体骨架基本信息 |
|
| 载体名称: | pMAK700, pMAK705, pBS-TS |
| 载体出品公司: | -- |
| 载体抗性: | 氯霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 8673 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 未知 |
| 生长条件: | 30 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | pKOV gene replacement vector,pKO5 |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | pK03-L: 5'-AGGGCAGGGTCGTTAAATAGC-3' |
| 3' 测序引物 | pK03-R: 5'-TTAATGCGCCGCTACAGGGCG-3' |
|
文献引用方法 |
|
| 材料和方法部分: | pKOV was a gift from George Church (Addgene plasmid # 25769 ; http://n2t.net/addgene:25769 ; RRID:Addgene_25769) |
| 参考文献部分: | Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. Link AJ, Phillips D, Church GM. J Bacteriol. 1997 Oct . 179(20):6228-37. PubMed 9335267 |
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 pKOV 8662 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pKOV
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8662)
AUTHORS Link AJ, Phillips D, Church GM
TITLE Methods for generating precise deletions and insertions in the
genome of wild-type Escherichia coli: application to open reading
frame characterization.
JOURNAL J Bacteriol. 1997 Oct . 179(20):6228-37.
PUBMED 9335267
REFERENCE 2 (bases 1 to 8662)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8662
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 118..220
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 221..880
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(287..306)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
primer_bind 3484..3501
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
rep_origin 4111..4565
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4197..4216)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4407..4428
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 4669..5114
/gene="sacR"
/label=sacB promoter
/note="sacB promoter and control region"
CDS 5115..6536
/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
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATVTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
CDS complement(6929..7879)
/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
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin 7927..8149
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
ORIGIN
1 ccctttcgtc ttcgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc
61 tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga
121 tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt
181 ttttgagtta tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
241 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
301 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
361 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
421 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
481 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
541 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
601 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
661 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
721 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
781 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
841 attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg
901 gtgcccttaa acgcctggtt gctacgcctg aataagtgat aataagcgga tgaatggcag
961 aaattcgaaa gcaaattcga cccggtcgtc ggttcagggc agggtcgtta aatagccgct
1021 tatgtctatt gctggtttaa tcggtacccg gggatcgcgg ccgccccgaa cccaactgat
1081 gcagtatccc gggcgctaca acatgatctc agtgatgtta tcggtcagga acaaggaaag
1141 cgaggactgg aaattaccgc tgctggcggg cacaaccttt tactgattgg gccgccggga
1201 acaggtaaaa caatgctcgc cagccgtatt aatggcctgt tgccagattt aagcaatgaa
1261 gaggcactgg agagtgctgc gatattaagt ctggtaaatg ctgaatcagt acaaaaacaa
1321 tggcggcagc gcccgttccg ctcacctcat cacagtgcat cgttaactgc gatggtaggc
1381 ggtggcgcaa ttccagggcc cggtgaaatt tcgctggcgc ataacggcgt gctttttctt
1441 gatgagctac ctgaatttga acggcgtaca ccggatgcct tgcgagagcc gattgaatcc
1501 gggcagatcc atctttcacg cacacgagca aaaataacct atccagcccg tttccagctt
1561 gttgcagcga tgaatcccag ccctaccggg cattatcagg gaaaccataa ccgctgcacg
1621 ccagaacaga cattacgtta tctcaaccgg ctctcggggc cctttctcga ccgcttcgat
1681 ctctcactgg agatcccatt accacccccc ggcactttga gtaaaacggt agtgccggga
1741 gaaagcagcg ccaccattaa acaacgcgta atggccgcca gagagcgcca atttaagcgg
1801 cagaataaac tgaatgcctg gctggatagt ccggaaatac gccaattctg caaacttgag
1861 agcgaagatg cgatgtggtt ggaaggaaca ctgatccatc tggggttatc gattcgtgcc
1921 tggcagcggt tattgaaagt tgcacgaacc attgctgata ttgatcagtc tgacattatc
1981 acacgtcagc atttgcagga ggcagttagc tatcgagcga ttgaccgttt gctcatccat
2041 ctgcagaaac tactgacata aaaaaagggc atttcgccct ttttattaat cgtcagaatc
2101 ggtgtagtct tcagcacctt caacctgcgg tttaccgcgg aaagggtgtg aaaacgtttt
2161 ggacgcttaa tacgcgtcat atacttggac cacacgcgtt ctgcttctgt cactggctca
2221 cgttcgccac ggcatactgc tacgaagagt ttctcttcct cggtaaccgg ctcgcgtttg
2281 ccaagatcca actcattgaa ggcataacca tgacgctcaa gcagttgtgc ctctttgatg
2341 gtgaaatcac catgacgaga gaatccacgt ggataatgtt tattgtcgaa atatcgatta
2401 gtcgtcgtaa agctttccgc catcctgcac gctcctaatt ctttgaccga gctagttatg
2461 gcgcggagta ttagttacgc ttgacagagt gtaaaacaaa acatttaaat cataacgaca
2521 aataattttg cggagagcac tgtggatgtt ataaatttgg agtgtgaagg ttattgcgtg
2581 tggtaaaaca gctgcatgaa cgccagcttg atcttcttat taccactgaa gcgcccaaaa
2641 tggacgaatt tagtagtcag ttgctgggat atttcacttt agcgctttat accagtgccc
2701 cttcaaaact aaagggagat cttaattatc tgcgacttga gtgggggcca gattttcaac
2761 agcatgaggc aggtttgatc ggtgctgacg aagtgcccat tctgacaacc agttctgctg
2821 aactggcaca gcaacagatt gcgatgctta atggttgcac ctggctaccc gtcagctggg
2881 cgcgtaaaaa aggcggcctg cataccgttg tcgatagcac aacactttca cggccgcttt
2941 atgccatatg gctgcaaaat agcgataaaa atgcgttgat tcgcgatctt ttgaaaatta
3001 acgtgctgga tgaagtgtat taatatgaat ggctggcaag gatgccggta gaaggattta
3061 cttcggagag ggttatttca gataaaaaaa atccttacgt ttcgctaagg atgatttctg
3121 gcaggggcgg agagactcga actcccaaca cccggttttg gagaccggtg ctctaccaat
3181 tgaactacgc ccctaattag ggtggcggaa cggacgggac tcgaacccgc gaccccctgc
3241 gtgacaggca ggtattctaa ccgactgaac taccgctcca ccgaattctt ttacaaccac
3301 cggttttatg accggcttac tgcttaattt gatgcctggc agttccctac tctcgcatgg
3361 ggagacccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca
3421 ggtgggacca ccgcgctacg gccgccaggc aaattctgtt tcatcagacc gcttctgcgt
3481 tctgatttaa tctgtatcag gctgaaaatc ttctctcatc cgccaaaaca tcttcggcgt
3541 tgtaaggtta agcctcacgg ttcattagta ccggttagct caacgcatcg ctgcgcttac
3601 acacccggcc tatcaacgtc gtcgtcttca acgttccttc aggactctca aggagtcagg
3661 gagaactcat ctcggggcaa gtttcgtgct tagatgcttt cagcacttat ctcttccgca
3721 tttagctacc gggcagtgcc attggcatga caacccgaac accagtgatg cgtccactcc
3781 ggtcctctcg tactaggagc agcccccctc agttctccag cgcccacggc agatagggac
3841 cgaactgtct cacgacgttc taaacccagc tcgcgtacca ctttaaatgg cgaacagcca
3901 tacccttggg acctacttca gctccaggat gtgatgagcc gacatcgagg tgccaaacac
3961 cgccgtcgat atgaactctt gggcggtatc agcctgttat ccccggagta ccttttatcc
4021 gttgagcgat ggcccttcca ttcagaacca ccggatcact atgacctgct ttcgcacctg
4081 ggatcctcta gagtcgaccg gagaatggcg cgcgccctgt agcggcgcat taagcgcggc
4141 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
4201 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
4261 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
4321 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
4381 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
4441 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
4501 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
4561 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
4621 atacattcaa atatgtatcc gctcatnnca ngatcctttt taacccatca catatacctg
4681 ccgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg tgattaaaaa
4741 ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa aagaaacaga
4801 tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt tctatcaaac
4861 aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg aggtcgaaaa
4921 gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg taaagggcaa
4981 agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg tgcgtaacta
5041 acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag tacataaaaa
5101 aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta
5161 ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc
5221 catataagga aacatacggc atttcccata ttacacgcca tgatatgctg caaatccctg
5281 aacagcaaaa aaatgaaaaa tatcaagttc ctgagttcga ttcgtccaca attaaaaata
5341 tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac gctgacggca
5401 ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat cctaaaaatg
5461 cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct attgacagct
5521 ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat gattctatcc
5581 taaaagacca aacacaagaa tggtcaggtt cagccacagt tacatctgac ggaaaaatcc
5641 gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg acaactgcac
5701 aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag gattataaat
5761 caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca
5821 actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa gataaaggcc
5881 acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa ggcgaagaat
5941 ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa
6001 aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct ctcggtatga
6061 ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt gcatctaaca
6121 cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa tggtacctgt
6181 tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac gatatttaca
6241 tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac aaaactggcc
6301 ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca cacttcgctg
6361 tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac agaggattct
6421 acgcagacaa acaatcaacg tttgcgccaa gcttcctgct gaacatcaaa ggcaagaaaa
6481 catctgttgt caaagacagc atccttgagc aaggacaatt aacagttaac aaataaaaac
6541 gcaaaagaaa atgccgatat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc
6601 ctgattcagg ctgtctatgt gtgactgttg agctgtaaca agttgtctca ggtgttcaat
6661 ttcatgttct agttgctttg ttttactggt ttcacctgtt ctgttaggtg ttacatgctg
6721 ttcatctgtt acattgtcga tctgttcatg gtgaacagct ttaaatgcac caaaaactcg
6781 taaaagctct gatgtatcta tcttttttac accgttttca tctgtgcata tggacagttt
6841 tccctttgat atctaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc
6901 actgatagat acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct
6961 ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatgcttac
7021 tttgcatgtc actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag
7081 catcgtgtag tgtttttctt agtccgttac gtaggtagga atctgatgta atggttgttg
7141 gtattttgtc accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt
7201 gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca
7261 ccaatttcat attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt
7321 taagcctttt aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa
7381 ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact
7441 cataaatcct catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta
7501 tgaatttttt taactggaaa agataaggca atatctcttc actaaaaact aattctaatt
7561 tttcgcttga gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag
7621 gattcctgat ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat
7681 aagcattttc cctactgatg ttcatcatct gaacgtattg gttataagtg aacgataccg
7741 tccgttcttt cctcgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa
7801 ttagcttggt ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga
7861 aaacaactaa ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat
7921 tgagatgggc tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta
7981 aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg
8041 ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga
8101 aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt
8161 cagttccgca gtattacaaa aggatgtcgt aaacgctgtt tgctcctcta caaaacagac
8221 cttaaaaccc taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc
8281 cttctgtctc cgaccatccg gcacctgagt cgctgtcttt ttcgtgacat tcagtccgct
8341 gcgctcacgg ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt
8401 catgcaagga aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt
8461 atggcgggtc tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct
8521 gattttccag tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc
8581 acccagtaag gcagcggtat catcaacagg cttacccgtc ttactgtcng gatcgacgct
8641 ctcccttatg cgactcctgc at
//
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