Clone: EvNO00061657
| Clone ID: | EvNO00061657 |
| Is Verified: | U |
| Status: | AVAILABLE |
| Source: | Mekalanos lab |
| Description: | Vector for transposon-based mutagenesis in bacteria, with arabinose-inducible promoter to drive expression of gene adjacent to transposon insert (modified promoter-out approach); kanamycin resistance; restriction enzyme cloning. |
| Comments: | This shipment includes the following items: 1) Purified pNJ17 (Judson and Mekalanos, 2000. PMID: 10888841). 2) E. coli strain DH5alpha lambda pir. 3) E. coli strain BW20767 (Metcalf, et al., 1996. PMID: 8693022). It is recommended that the following approaches be used for handling this plasmid: 1) Avoid single colony isolation for strains containing pNJ17. Instead, it is preferable to scrape up multiple individual colonies from a plate for establishing stocks and cultures. Strain passage should be minimized. This is because the transposon hops out at a relatively high frequency, causing plasmid loss. Plasmid loss cannot be tracked by kanamycin resistance because insertion of the transposon into the host strain chromosome should also cause a resistant phenotype. Please also keep in mind that mariner-containing plasmids can be prone to spontaneous rearrangement. 2) DH5alpha lambda pir is suitable for cloning or plasmid maintenance, but it is not competent to mobilize the plasmid into recipient strains. For mating, the plasmid should be introduced into BW20767 or another mating strain of your choice. Multi-colony isolation should also be used for the mating strains once the plasmid has been introduced. |
| Type: | No insert |
| Verification Method: | |
| Distribution: | Academic and non-profit labs |
| Special MTA: |
| Map: | pNJ17.pdf |
Related Identifiers:
| Original Clone ID | pNJ17 |
| SYNONYM | TnAraOut |
Sequencing Information:
| Phred Score: | 662 |
| Date: | 2018-04-23 |
Last Passing Sequencing Read:
>29051-13_EvNO00061657-1-M13F_E2 662 1512 48 882 NNCCCACGAGCTATAGGCGATGAAGGACCGGTCTAACAGACAAAACATTT TTTTGTGAAAATTCGTTTTTATTATTCAACATAGTTCCCTTCAAGAGCGA TACAACGATTATAACGACCTTCCAATTTTTTGATACCATTTTGGTAGGAC TCCTTCGGGTTTGCCTCAAAATATGCCTCAGTTTCGGCGATCACCTCTTC ATTGCAGCCAAATTTTTTCCCTGCGAGCATCCTTTTGAGGTCTGAGAACA AGAAAAAGTCGCTGGGGGCCAGATCTGGAGAATACGGCGGGTGGGGAAGC AATTCTAATCCCAATTCATGAATTTTTGCCATCGTTCTCAATGACTTGTG GCACGGTGCGATGTCTTGGTGGAACAACACTTTTTTCTTCTTCATGTGGG GCCGTTTTGCCGCGATTTCTACCTTCAAACGCTCCAATAACGCCATATAA TAGTCACTGTTGATGGTTTTTCCCTTCTCAAGATAATCGATAAAAATTAT TCCATGCGCATCCCAAAAAACAGAGGCCATTACTTTGCCAGCGGACTTTT GAGTCTTTCCACGCTTCGGAGACGGTTCACCGGTCGCTGTCCACTCAGCC TACTGTCGATTGGACTCACGAGTGTAGTGATGGAGCCATGTTTCATCCAT TGTCACATATCGACCGAAAAACTCGGGTGTATTACGAGTTAACAGCTGCG AACACCGCTCATAATCATCAACACGTTGTTGTTTTTGCTCAAATGTGAGC TCGCGCGACACCCATTTTGCACAGAGCTTCCGCATATCCAAATATTGATG AATGATATGACCAACACGTTCCTTTGATATCTTTAAGGCCTCTGCTATCT CGATCAACTTCATTTTACCGTCATTCAAAATCATTTTGAGGATTTTTATG ATGTTTTCGTCGGTAACCACCTCTTTCCGGCGTCCACTGCGTTTCACAGT CCTCGTGCTCATTTCACACGCTTGAATTTTGCATACCACCATTATTGTTG ATTCCCTGGGGCAGAGCCCGAAACTCTTTTCAACCAAGTTTTGCTTCACT GGATTTTTCCCCTTCGAAACAGATTTTATCAAACACAAATCCTTTTTTTT CCAAGTTTCCAATAACAAAGTTGTTTTAAAAAACCCTTTTTCCAAATATT GGCTTACAAGTCAATTTGAACCAATCTTTAAGTTGGACTATAAAAAAAAA GCTTTAAACTCCACCCTTATGGCCTCTCCACGGCGCGGAACTCCCTAAAA TTTTGGGGCAGGCCAAAGCTACTGAACCAGGGCCCCATTAATTTCGAAAC CCAAGTGGAAAAGAAAAAAAGGGGCTGGGCGGATGGCCTCCCAACCCCCC CTTAGCCCAGGCTTCCCACAATAAAGAGGCAATTCCCCCAGAACCCCAAG GAACCCAAAATTTATTTAATCAGACCCCCCCCCATTGGNTATTTNNNGGN CCCTTCCCCTCTACTATACATTNNNNNNNNNNNNNNNNNNNNNNNNNNNN
Vector Information:
| Vector Name: | pNJ17 |
| Type: | bacterial plasmid |
| Description: | Vector for transposon-based mutagenesis in bacteria, with arabinose-inducible promoter to drive expression of gene adjacent to transposon insert (modified promoter-out approach); kanamycin resistance; restriction enzyme cloning. |
| Properties: | inducible expression, mutagenesis vector, transposon mediated gene disruption |
| Comments: | This shipment includes the following items: 1) Purified pNJ17 (Judson and Mekalanos, 2000. PMID: 10888841). 2) E. coli strain DH5alpha lambda pir. 3) E. coli strain BW20767 (Metcalf, et al., 1996. PMID: 8693022). It is recommended that the following approaches be used for handling this plasmid: 1) Avoid single colony isolation for strains containing pNJ17. Instead, it is preferable to scrape up multiple individual colonies from a plate for establishing stocks and cultures. Strain passage should be minimized. This is because the transposon hops out at a relatively high frequency, causing plasmid loss. Plasmid loss cannot be tracked by kanamycin resistance because insertion of the transposon into the host strain chromosome should also cause a resistant phenotype. Please also keep in mind that mariner-containing plasmids can be prone to spontaneous rearrangement. 2) DH5alpha lambda pir is suitable for cloning or plasmid maintenance, but it is not competent to mobilize the plasmid into recipient strains. For mating, the plasmid should be introduced into BW20767 or another mating strain of your choice. Multi-colony isolation should also be used for the mating strains once the plasmid has been introduced. |
| Map: | pNJ17.pdf |
| Sequence: | pNJ17.txt |
| Size (bp): | 6722 |
| Form: | dsDNA |
Host Information:
| Host Strain | Is Used In Distribution | Description |
| DH5-alpha lambda pir | Y | clone provided in DH5-alpha lambda pir (Mekalanos lab) |
Antibiotic Selections:
| Host Type | Marker |
| bacterial | kanamycin |
Recommended Growth Condition:
| Host Type | Selection Condition | Growth Condition | Comments |
| bacterial | 50ug/mL kanamycin | Growth with the single antibiotic in LB at 37 degrees is recommended. | Conditions for Gateway-type vectors in recombined (with insert) form and other kanamycin resistant vectors. |
Authors:
| Author Name | Author Type |
| John Mekalanos | Academic researcher, PI |
| Su Chiang | Academic researcher, clone donor |
| Nicholas Judson | Academid researcher, clone first author |
Publications:
| PMID | Title |
| 10888841 | TnAraOut, a transposon-based approach to identify and characterize essential bacterial genes |
