pSGKP-spe Plasmid
PVTY01216 pSGKP-spe (Alias No.117234) is a CRISPR gene knockout empty vector designed for targeted gene disruption in Escherichia coli. This high-copy-number plasmid features the J23119 constitutive promoter for robust guide RNA expression, spectinomycin prokaryotic resistance via the aadA gene for reliable bacterial selection, and a pUC replicon backbone providing 500-700 copies per cell for superior plasmid yield. Optimized for transformation into E.coli DH5α competent cells at 37°C, this streamlined backbone supports seamless cloning of sgRNA cassettes for CRISPR-Cas9 mediated gene knockout applications without additional eukaryotic screening markers.
Ordering Information
| Catalog No. | Product Name | Size | Form | Availability |
|---|
| PVTY01216 | pSGKP-spe Plasmid | ~4,705 bp / 2μg | Lyophilized powder | In Stock |
Technical Specifications & Vector Traits
| Vector Type | CRISPR Gene Knockout Empty Vector | Fragment Type | CRISPR |
| Promoter | J23119 (constitutive bacterial promoter) | Prokaryotic Resistance | Spectinomycin (Spe) |
| Replicon | pUC (high-copy) | Copy Number | High (~500-700 copies/cell) |
| Host Organism | E.coli DH5α | Culture Temperature | 37°Celsius |
| Alias/Clone ID | No.117234 |
| Applications | Gene knockout, genome engineering, CRISPR-Cas9 mediated gene disruption, sgRNA expression |
| Species Origin | Empty vector backbone (no species-specific insert) |
Key Features & Benefits
- J23119 Constitutive Promoter: Strong, well-characterized Anderson series promoter ensures consistent gRNA expression without requiring external induction or IPTG throughout all growth phases
- Spectinomycin Resistance: Single-marker selection system using aadA aminoglycoside adenyltransferase gene provides robust selection pressure at standard concentrations (50-100 μg/mL) while maintaining compatibility with Kan/Amp/Rif co-transformation strategies
- pUC High-Copy Replicon: ColE1-derived origin provides 500-700 copies per cell for superior plasmid yield during maxiprep preparation and enhanced sgRNA expression levels for improved editing efficiency
- Empty Vector Flexibility: Ready-to-use backbone allows rapid Golden Gate or restriction enzyme-based cloning of custom sgRNA sequences for any target gene of interest
- Optimized for E.coli: Validated performance in DH5α competent cells at standard 37°C culture conditions with reliable transformation efficiency (>10^8 cfu/μg DNA)
- CRISPR-Ready Architecture: Pre-configured with gRNA scaffold and terminator sequences downstream of J23119 promoter for immediate sgRNA cassette insertion via BsaI/BbsI sites
- Cross-Compatible Design: Spectinomycin marker allows co-transformation with kanamycin, ampicillin, or rifampicin plasmids carrying Cas9 expression cassettes without selection conflict
Recommended Applications
- Bacterial Gene Knockout: Construct sgRNA expression cassettes targeting essential or non-essential E.coli genes for functional genomics studies via CRISPR-Cas9 mediated double-strand break induction and NHEJ repair pathway exploitation
- Multiplexed Gene Disruption: Clone multiple sgRNA expression units in tandem for simultaneous targeting of several genomic loci in a single transformation event to study synthetic lethal interactions
- Allelic Replacement: Combine with donor DNA templates containing homology arms for precise gene replacement or scarless point mutation introduction through homology-directed repair (HDR)
- Library Construction: Use as backbone for pooled sgRNA library generation to screen genome-wide loss-of-function phenotypes in bacterial populations under selective conditions
- Synthetic Biology Prototyping: Rapid assembly of CRISPR regulatory circuits using standardized modular cloning strategies compatible with BioBrick/Golden Gate standards for metabolic engineering applications
Handling Guidelines & Cautions:
- This product is FOR RESEARCH USE ONLY! Not intended for diagnostic, therapeutic, or human/animal use.
- The item is supplied as lyophilized powder. Centrifuge at 5000 rpm/min for 1 min before opening. Add 20 μl ddH2O into the tube to reconstitute the plasmid.
- Take 2 μl reconstituted plasmid into 100 μl corresponding competent cell (DH5α), centrifuge briefly, then spread fully onto LB+Spe plate.
- Shipping temperature is 2–8 degrees centigrade (℃). Store lyophilized plasmid at −20°C upon receipt.
- For optimal transformation efficiency, use high-quality chemically competent or electrocompetent E.coli DH5α cells (>10^8 cfu/μg DNA).
- When designing sgRNA targets, ensure NGG PAM sequence compatibility with your chosen Cas9 ortholog (typically SpCas9). Avoid off-target sites with ≤3 mismatches in seed region.
- Verify insert orientation and sequence integrity by colony PCR and Sanger sequencing before proceeding to editing experiments.
▶ Click to expand/collapse pSGKP-spe Sequence (~4,705 bp)
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGACGCGTATTGGGATGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATACGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGGAGTCAGTGAACAGATACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGTTTGTTTGCCGTAATGTTTACCGGAGAAATCAGTGTAGAATAAACGGATTTTTCCGTCAGATGTAAATGTGGCTGAACCTGACCATTCTTGTGTTTGGTCTTTTAGGATAGAATCATTTGCATCGAATTTGTCGCTGTCTTTAAAGACGCGGCCAGCGTTTTTCCAGCTGTCAATAGAAGTTTCGCCGACTTTTTGATAGAACATGTAAATCGATGTGTCATCCGCATTTTTAGGATCTCCGGCTAATGCAAAGACGATGTGGTAGCCGTGATAGTTTGCGACAGTGCCGTCAGCGTTTTGTAATGGCCAGCTGTCCCAAACGTCCAGGCCTTTTGCAGAAGAGATATTTTTAATTGTGGACGAATCGAACTCAGGAACTTGATATTTTTCATTTTTTTGCTGTTCAGGGATTTGCAGCATATCATGGCGTGTAATATGGGAAATGCCGTATGTTTCCTTATATGGCTTTTGGTTCGTTTCTTTCGCAAACGCTTGAGTTGCGCCTCCTGCCAGCAGTGCGGTAGTAAAGGTTAATACTGTTGCTTGTTTTGCAAACTTTTTGATGTTCATCGTTCATGTCTCCTTTTTTATGTACTGTGTTAGCGGTCTGCTTCTTCCAGCCCTCCTGTTTGAAGATGGCAAGTTAGTTACGCACAATAAAAAAAGACCTAAAATATGTAAGGGGTGACGCCAAAGTATACACTTTGCCCTTTACACATTTTAGGTCTTGCCTGCTTTATCAGTAACAAACCCGCGCGATTTACTTTTCGACCTCATTCTATTAGACTCTCGTTTGGATTGCAACTGGTCTATTTTCCTCTTTTGTTTGATAGAAAATCATAAAAGGATTTGCAGACTACGGGCCTAAAGAACTAAAAAATCTATCTGTTTCTTTTCATTCTCTGTATTTTTTATAGTTTCTGTTGCATGGGCATAAAGTTGCAAGCTTGATATCTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTCGAGACCATTGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCATCCCAATGGCGCGCCGAGCTTGGCTCGAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCCCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
References
- Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096. doi:10.1126/science.1258096. PMID: 25430718.
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823. doi:10.1126/science.1231143. PMID: 23287718.
- Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-1278. doi:10.1016/j.cell.2014.05.010. PMID: 24906146.
- Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. doi:10.1038/nprot.2013.143. PMID: 24157548.
- Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013;31(3):233-239. doi:10.1038/nbt.2508. PMID: 23360777.
- Knight SC, Manikantan L, Hoel CM, Wilson C, Stainier DYR. Efficient CRISPR-Cas9 genome editing in Escherichia coli using a simplified single-plasmid system. G3 (Bethesda). 2020;10(8):2653-2662. doi:10.1534/g3.120.401382. PMID: 32554487.
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Frequently Asked Questions (FAQ)
Q1: What is the difference between pSGKP-spe and other pSGKP variants?
pSGKP-spe uses spectinomycin (aadA) as its sole antibiotic resistance marker, distinguishing it from pSGKP-km (kanamycin), pSGKP-Str (streptomycin), and pSGKP-Rif (rifampicin) variants. This makes it ideal when your host strain already carries resistance to Kan, Amp, Str, or Rif markers, enabling clean dual-plasmid systems where one plasmid carries Cas9 (with a different antibiotic marker) and pSGKP-spe carries the sgRNA cassette.
Q2: What does alias "No.117234" mean?
No.117234 is an internal clone identifier assigned during the original construction of this plasmid. This alias can be used as an alternative reference when searching inventory databases or cross-referencing with internal lab records. The primary catalog number for ordering remains PVTY01216. When citing this plasmid in publications, use the catalog number PVTY01216 as the official identifier.
Q3: How do I clone my sgRNA into this vector?
Design complementary oligonucleotides encoding your 20-nt target sequence flanked by BsaI/BbsI overhangs compatible with the gRNA scaffold insertion site downstream of J23119. Anneal oligos, phosphorylate if needed, ligate into BsaI-linearized vector, transform into DH5α, select on LB+Spe plates (50-100 μg/mL), verify by colony PCR using primers binding within the promoter region followed by Sanger sequencing confirmation.
Q4: Can this plasmid express Cas9?
No, pSGKP-spe is an sgRNA expression-only vector. For complete CRISPR editing, you need a separate Cas9 source: either a co-transformed plasmid (e.g., pCas9 with different antibiotic marker like Amp, Chl, or Kan) or a chromosomal Cas9 integration strain such as E.coli HMS174(DE3)pLysS with inducible T7-driven Cas9 expression. The pSGKP family is specifically designed for guide RNA delivery only.
Q5: What spectinomycin concentration should I use?
Standard spectinomycin selection for E.coli DH5α carrying pSGKP-spe is 50-100 μg/mL in LB agar plates. Prepare stock solution (50 mg/mL in water, filter-sterilized) and add to cooled autoclaved media (<55°C). Some strains may require higher concentrations up to 150 μg/mL depending on copy number and promoter strength. Spectinomycin is stable at 37°C for extended incubation periods.
Q6: What is the typical editing efficiency?
Editing efficiency varies by locus but typically ranges from 30-80% for gene deletions and 15-50% for point mutations in E.coli using pSGKP-based systems combined with Cas9. Key factors affecting efficiency include: sgRNA design quality (avoid off-targets, target near origin of replication), donor template format (double-stranded DNA with 500-1000 bp homology arms outperforms single-stranded oligos), strain genotype (mutS deficiency improves HDR), and transformation method (electroporation preferred over chemical competence).
Q7: How do I cure the plasmid after editing?
Since pSGKP-spe lacks temperature-sensitive elements, curing requires serial passage without antibiotic pressure: grow edited clones in non-selective LB broth at 37°C for 3-5 passages with 1:1000 dilution each time, plate on non-selective agar, replica-plate colonies onto LB+Spe to identify spectinomycin-sensitive clones, and confirm plasmid loss by PCR for vector backbone sequences. This yields clean, marker-free edited strains suitable for downstream characterization.
