Kanamycin resistance cassette sequence Insert a functional copy of the gene ectopically at an unlinked chromosomal locus between the lctP and aspC genes under control of lac regulatory sequences, and linked to an Erythromycin-resistance cassette. fgb. Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. Similar results were obtained for the agronomically important plant species canola (Brassica napus) [1], indicating that the Arabidopsis resistance gene represents a new robust selectable marker for plant transformation. 2 kanR cassette from Dr. From the resulting plasmid, pKOK5, the lacZ-KmR cassette was recloned by means of BamHI into plasmid pKOK4, a mobilizable d … The difference between this plasmid and pKDH (Addgene plasmid # 67584) is that pSIM24 KanR has a kanamycin resistance cassette where pKDH has a hygromycin resistance cassette. denticola 35405 and demonstrated the utility of a kanamycin resistance cassette in the mutagenesis of T. plasmid. Peter Nixon's lab contains the insert Kanamycin resistance gene and is published in Commun Biol. kanMX selector module conferring kanamycin resistance, for gene disruption in yeast, kanMX6 version. A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. 2011. Day 1: Transform recombinase plasmid . To generate the pRHB. Between the kanamycin and hphMX4 resistance genes there is a preferential cloning site that allows the use of both KpnI and BglII restriction enzymes. g. , strain LF3, Fig. coli through expression of kanamycin resistance cassette (Kan R). This method differs in two significant ways from traditional methods of linker mutagenesis. (2015) revealed a point mutation exactly at the same location inside the kanamycin resistance cassette in both the spontaneous nif positive mutants. The kanamycin resistance gene is oriented for expression of downstream genes (Figure 8A). Tip After recombination into a tumor inducing plasmid delivery plasmid, this cassette confers selectable kanamycin resistance to transformed sunflower cells. Resulting primary transformants of pKAN B and pKANα B displayed two distinctive colony types, as shown in Figure 3, after 48 h of growth at 30 °C Cre-Kan cassette contains the 34 bp lox71 sequence, the Cre expression cassette, the kanamycin resistance cassette and the 34 bp lox66 sequence. Dec 14, 1989 · A cassette similar to the one described here is intended to yield translational, not transcriptional, fusions (Tiedeman and Smith, 1988). Further, the mutants were constructed by use of a resistance cassette that can be easily eliminated (Datsenko and Wanner, 2000). To overcome this limitation, we exchanged the chloramphenicol acetyl transferase in both cases for either the MLS (macrolide-lincosamide-streptogramin B) or kanamycin resistance cassette. The transcription start site was identified by high-resolution S1 mapping and the kmr mRNA was shown to have a long leader of about 400 bp. , 1994). Strategies employed for cloning a kanamycin resistance cassette (kanR) via selectable marker restriction enzyme mediated gene disruption. Aug 16, 2012 · The novel T7C p/p system created was based on an expression cassette (ECKm cassette) (Figure 1) controlled by a T7 promoter introduced into the expression vector. There are several versions of cassette plasmids containing this kan-a mycin phosphotransferase gene from Tn903[e. Figure 3. 5 µg/ml) and kanamycin (50 µg/ml) resistance upon E. The expression cassette can be liberated by NotI digestion. 2020 May 7;3(1):215. Feb 23, 2023 · Here, we used Cre to induce expression of four antibiotic resistance genes, which we selected for their ubiquity in synthetic biology applications as well as their range of mechanisms of action May 3, 2017 · Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Use text editor or plasmid mapping software to view sequence. pCAT vectors transform C. mammalian tissues because the transgene expression cassette is silenced. Plasmid for yeast gene deletion using the kanMX selectable marker conferring kanamycin resistance. The kanamycin resistance cassette was flanked by FRT sites, and, thus, was flipped out using the site-specific recombinase Flp provided by the plasmid pCP20. Com-pared to the widely used ermF-ermAMcassette, the kanamycin cassette used in the transformation experiments gave rise to addi-tional antibiotic-resistant T. Bacterial vector encoding an N-terminal 6xHis-TEV cassette and kanamycin resistance plus a SacB negative selection marker, for purification of recombinant proteins. Here, we show that transcriptional silencing can be Apr 5, 1981 · Nucleotide sequence of the kanamycin resistance transposon Tn903 J Mol Biol. Make electrocompetent cells of the desired E. Mar 19, 1999 · promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. Jan 1, 1995 · REFERENCES Alexeyev, M. necator low electroporation transformation efficiency. coli and Agrobacterium tumefaciens. Subsequent excision of the KmR cassette with ApaI and religation of the plasmid leaves behind six base pairs (5'-GGGCCC) that result in the insertion of two amino acids in … Apr 20, 2016 · Cloning: I insert the promoter sequence upstream of the kanamycin resistance gene in the plasmid vector to ensure it drives the expression of the antibiotic resistance marker. besciiwas sensitive to kanamycin at 10 g·ml1. 1a). It lacks a terminator. To our knowledge, no other resistance cassette exists which allows for the combi- nation of insertion mutagenesis with the possibility of monitoring promoter activity by means of the lacZ gene. 2011; 65(3):246–52. The 1. 1 B). May 3, 2017 · The plasmid backbone contained in DNA vectors silences expression of the transgene. Use with SnapGene software or the free Viewer to visualize additional data and align other sequences. pastoris. 6. Dec 1, 2012 · This leads to loss of both the kanamycin resistance cassette and the wild-type allele of rpsL, and cells become Kan s Strp r. 4. Promoter and terminator regions are indicated by full boxes, and the LIP2 prepro (LIP2pp) by an open box. This set of . Proc Oct 18, 2017 · All transductants were verified by PCR to ensure the presence of the kanamycin cassette. Base sequence determination by Barney et al. Also known as pFA6a-kanMX4. 1016/j. Barany, F. 2. In Shank3 gKO mice, only a 130-bp sequence containing Frt and loxP sites was identified (Figure 3A, lower panel). In this study, the 6. Kanamycin resistance cassette including promoter and coding sequence in the reverse orientation. 04. bescii. K3 (Elhai and Wolk CP, 1988, Gene 68: 119-138). Plasmid carrying the kanMX selector module conferring kanamycin resistance. This plasmid confers tetracycline (12. Only TetA is required for tetracycline resistance. aphA-3. Plasmid. Use strain TT23691 as template for this cassette. . Oct 11, 2023 · As a general overview, we can see the known trends in resistance where aacAs confer resistance against kanamycin and tobramycin; aacCs against gentamicin; aadAs against streptomycin; aadB against Kanamycin resistance protein. High-fidelity DNA polymerase master mix for PCR cloning. 2 kb of RD to generate the MC. However, since all currently used antibiotic-resistance genes are of bacterial origin, there ha … Jan 24, 2024 · Successful recombination enables survival of E. pUC4K Is a vector that carries the kanamycin resistance determinant from Tn903 (1) and has been used not only in the construction of new A 15-nucleotide synthetic UTR (the so-called universal translational enhancer [UTE]) was placed upstream of a kanamycin/neomycin phosphotransferase (kanaR) gene to create a novel expression cassette, UTE-kanaR. pUC. The second antibiotic-resistance gene cassette to be modified was the kanamycin-resistance determinant from Tn903. coli has been replaced by a kanamycin-resistance (KanR) cassette flanked on each Agrobacterium binary vector for plant transformation, with hygromycin- and kanamycin-resistance and LacZ-GUS genes plus the pUC9 MCS. DNA Protein Eng. coli and G418 in P. We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification The resulting plasmid was named pMM and it contained genetic determinants conferring simultaneous resistance to ampicillin, kanamycin (geneticin for yeast) and hygromycin B. The modified kanamycin resistance gene allows for selection of transformants with kanamycin in E. 1038/s42003-020-0910-8. Cloning vector with a kanamycin resistance marker, suitable for generating ExoIII deletions. 358. Apr 21, 2014 · This led to the development, in 1988, of a kanamycin resistance cassette for use in gene disruption experiments . It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. Finally, to ensure Homologous sequence among these isolates immediately adjacent to the resistance gene was considered an Antibiotic Resistance Cassette (ARC). The polylinker came, in large part, from the plC20R polylinker (Marsh et al. : Procedures for linker insertion mutagenesis and use of new kanamycin resistance cassettes. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1,000 base pairs . MUSSO and Tara Hodam Department of Biological Sciences, University of South Carolina, Columbia, S C 29208 (U. Caius M. Made by exact replacement of camR coding region of uni-cmr-cassette with the coding region of gene aphA-2, encoding kanamycin resistance. To insert the loxP-STOP-loxP cassette, build a construct with 5′ targeting and 3′ targeting arms to direct integration by homologous recombination at the desired location in the mutated retrieved locus. In biology, a gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Be mindful of the orientation of the drug cassette with respect to the gene. To insert loxP bounded antibiotic resistance gene: The 1. Explore the Scientific R&D Platform Scientific intelligence platform for AI-powered data management and workflow automation Plasmid pRGD-SmR from Dr. Include a Frt-Neo-Frt cassette, which confers kanamycin resistance to bacteria for selecting desired clones. May 3, 2017 · The same restriction enzyme was used to insert a second copy of 2. We screened a variety of antibiotic resistance cassettes and found a kanamycin resistance gene that is a reliable selective marker for T. These can be used to provide a selectable genetic marker or to mobilize restriction sites and sense or nonsense codons into genes. As drawn in the map, this scar has an id Dec 9, 2009 · We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. These strains showed resistance to kanamycin at concentrations >50 g·ml1, whereas wild-type C. 1 plasmid containing kanamycin resistance cassette. ) Received The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The gene had a partial nucleotide sequence of 968 bp Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene (kanr), which confers resistance to G418 (Wach et al. 3-kb HincII fragment bearing the kanamycin resistance cassette from pUC4K (Vieira, 1982) was inserted into pLox1 (Palmeros, 2000), which had been cut with XbaI and blunted, to create pCM161. Keep in mind that for Step 6, it is helpful if the drug cassette is a size different from that of the replaced gene. The kanamycin and hygromycin resistance cassettes are used for selection in plants. Nov 25, 2021 · Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. kanMX selector module conferring kanamycin resistance, for gene disruption in yeast. Dec 7, 1989 · We demonstrate how a kanamycin-resistance (KmR) cassette flanked by polylinkers with multiple restriction sites can be used to introduce nucleotide (nt) sequence replacements into a region of interest. doi: 10. 3' entry vector containing FRT-flanked kanamycin resistance cassette (KanR) with pA for FLP Dec 14, 1989 · A lacZ gene without a promoter, but containing its ribosome-binding site, was cloned next to the kanamycin-resistance (KmR) gene of plasmid pUC4K, yielding a lacZ-KmR cassette. Jul 22, 2022 · Chen CY, Strobaugh TP Jr, Lindsey RL, Frye JG, Uhlich G. This cassette consists of the SV40 early promoter (P. Sep 7, 2010 · Plasmid pSP1 containing the functional ccdA cassette, the cer fragment and an almost completely truncated kanamycin resistance gene. can be replaced directly with the drug resistance. Explore the Scientific R&D Platform Scientific intelligence platform for AI-powered data management and workflow automation Jun 8, 2018 · To permit selection for kanamycin-resistant transformants of recA library plasmids, the kanamycin-resistance cassette was first removed from ∆recA::FRT-Kan R-FRT Keio strain JW2669 by expression Mammalian vector encoding a neomycin resistance gene, to select for stable integration of a cotransfected expression plasmid. Sequence analysis of these two kanamycin resistance cassettes revealed that they share 35% identity and 52% similarity at the amino acid sequence level; however, only the aphA2 cassette was able to confer resistance to kanamycin in T. Of 4288 genes targeted, mutants were obtained for 3985. orf. denticola genes. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. BioTechniques 18 (1995 ) 52-56. ΔGeneX represents the targeted gene to be deleted. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. Aug 11, 2017 · Cre-Kan cassette contains the 34 bp lox71 sequence, the Cre expression cassette, the kanamycin resistance cassette and the 34 bp lox66 sequence. bescii. This mutant library has proven to be incredibly useful in the fields of bacteriology, chemic … Sequence analysis of these two kanamycin resistance cassettes revealed that they share 35% identity and 52% similarity at the amino acid sequence level; however, only the aphA2 cassette was able to confer resistance to kanamycin in T. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. 2015 Jun;79:102-9. J-Kan vector, the pRHB was digested by SacI and XbaI to remove the wild-type kanamycin resistance gene sequence, followed by ligating to the SacI-XbaI double-digested origin for single-stranded DNA production. Jan 11, 1988 · A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K Nucleic Acids Res . F. Plasmid pIP1433. 6-kb pSNC3-Kan Plasmid sequence and annotations. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. tation of the chloramphenicol-resistance cassette in subsequent experiments. orf . The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. coli strain. Explore the Scientific R&D Platform Scientific intelligence platform for AI-powered data management and workflow automation The hygromycin resistance cassette in pMyC was replaced by a kanamycin resistance cassette using Gibson cloning. denticola, suggesting that a specific sequence is required in T. The target sequence and bridge RNA are separated by a DNA barcode of 12 nt (BC). Oct 25, 2013 · Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. 4. Plasmid pJet1. However, I cannot find the promoter region of this gene on the vector map. 3B). 1988 Jan 11;16(1):358. Gene. All primers are listed in Supplementary Table S3. To generate pMQ352, we amplified the LEU2 gene from pRS415 [ 31 ] with primers 1295 and 1298. Using the spectinomycin cassette to knock out genes can be tricky, with the kanMX selector module conferring kanamycin resistance, for gene disruption in yeast, kanMX2 version. b Restriction sites present on both sides of the cassette are boldface. Oct 12, 2016 · Preliminary genetic experiments 1 indicated that the kanamycin resistance trait of transducing phage whose AR gene segment was from JR72 (encoding NPTI), represented by ‘λbb kan-3’, is Dec 21, 1989 · We have developed plasmids with the Tn5 kanamycin-resistance gene (kan) flanked either symmetrically or asymmetrically by several restriction sites. 2) We replaced the kanamycin resistance cassette of pDOC-K with a lacZα drop-out cassette flanked by BsaI restriction sites to create a Golden thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of ei-ther replicating or integrating vector constructs in C. pKD3 and pKD4 are identical except for the region between the FRT sites, so they create an identical scar that has stop codons in all six reading frames. Feb 21, 2019 · Introduced in 2006, the Keio collection consisted of 3985 single-gene deletion mutants for nonessential genes in E. A synthetic polyadenylation signal terminates the kanamycin/neomycin expression. Dec 29, 1995 · The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. The WT donor and WT donor-binding loop are used. Transform with pCP20. The gentamicin resistance cassette is derived from Jan 10, 2021 · In terms of the inserted sequence in the targeted allele, an approximately 1. gentamicin resistance cassette aph(2) Gentamicin 25 ug/ml: 1089: BBa_P1013: gentamycin resistance cassette (aacC1 gene) 841: BBa_P1012: gentamycin/kanamycin resistance cassette 1943: BBa_P1003: kanamycin resistance cassette 967: BBa_K292003: Kanamycin resistance cassette and a double terminator 1070: BBa_K1642003: Kanamycin Resistance Cassette [30-150bp sequence fragment from upstream to BLA promoter from pkD46 ]-[Kan resistance region from a plasmid, starting with promoter ending with ORF stop]-[30-150 bp sequence starting from the Nov 9, 2010 · Although the kanamycin resistance cassette cannot be used in clinical strains (all the clinical strains of A. denticola colonies. 04085-15. In recent years the availability of genome sequence information has grown Jul 14, 2022 · Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. Feb 21, 2006 · In most cases, the coding region from the 2nd through the 7th codon from the C‐terminus has been deleted. coli). 7 kb PstI fragment of pKOK6 [20], which contains a promoterless lacZ-kanamycin resistance gene cassette, into the PstI site of the EZ::TN pMOD-3 Plasmid pC-G418-YR from Dr. A neomycin-resistance cassette (Neo. kanamycin resistance cassette. This plasmid is available through Addgene. 1016/0022-2836(81)90438-1. J-Kan vector, the pRHB was digested by SacI and XbaI to remove the wild-type kanamycin resistance gene sequence, followed by ligating to the SacI-XbaI double-digested Jul 7, 2016 · To expand the C. Oct 5, 2016 · The efficiency of integration of the kanamycin resistance cassette (approximately 2 kb) into the fliK locus of the strain BL21-DE3 was high and comparable to that observed in the strain K12 MG1655 (Fig. Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars. coli strains. By increasing the An/Tn content (nucleosome-relaxation sequences) in the ampicillin/kanamycin resistance genes, Kay and colleagues have achieved persistent plasmid-mediated transgene expression from a mouse liver. Dec 21, 1989 · Gene, 85 (1989) 205-207 Eisevier GENE 03306 205 Construction and characterization of versatile kanamycin-resistance cassettes derived from the Tn5 transposon* (Recombinant DNA; genetic markers; restriction site mobilization; translation terminators; polarity) Richard E. Used to create strains to complement mutations using the NICS system (Mehr and Seifert, 1998; Mehr, 2000). SV40 e), the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. , p34-Km (2), pUC4KS , Nov 1, 2020 · The mCloverBlaster sequence is introduced into the chromosome using lambda red recombineering, expression of I-SceI creates a double stranded break in the kanamycin resistance cassette that initiates a recombination event that can occur in the mClover3 repeats. Explore the Scientific R&D Platform Scientific intelligence platform for AI-powered data management and workflow automation Although this step is not required prior to injection, removing the kanamycin resistance cassette generates a nearly “seamless” recombinant BAC in which the fluorescent protein sequence is located in a genomic context that is almost identical to the endogenous gene of interest. During data processing, the largest number of reads were discarded between the Ktag1 and Ktag2 pattern matching steps (Table 1): ‘Ktag1’ corresponds with the sequence of the K2F forward primer, while ‘Ktag2’ corresponds with the sequence of the kanamycin resistance cassette immediately downstream from the K2F primer binding site (Fig a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. S. These sites were The neomycin/kanamycin determinant in pRL278 was derived from cassette C. Jul 1, 1996 · The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT. Status. Com- DNA sequence validation of various plasmid constructs were performed Dec 29, 1995 · The Spa cassette was originally a blunted fragment (nt 1-1158 in GenBaok sequence M69221) cloned in the Smal site of pBluescript 11 SK+ (Stratagene). 4kb RD vector. , 1987) or phosphinotricine phosphotransferase (from 35SpBARn, LeClere and Bartel, 2001) for kanamycin and BASTA selection, respectively. Amino acids. Characteristics of P. Consequently, a series of modular minimal plasmids, named pCAT, were constructed. Hans-Martin Fischer's lab contains the insert aadA and is published in Appl Environ Microbiol. The kan cassette originally came from pCP15. 2016 Feb 26. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the h … Aug 1, 2002 · The gene conferring kanamycin resistance (KanR) and the URA3 gene are indicated by an arrow. pCD33_Exon1-7_plus introns_C_Extended pY This led to the development, in 1988, of a kanamycin resistance cassette for use in gene disruption experiments . Rommens, in Trends in Plant Science, 2006. 1. Nucleotide sequence at the insertion sites of a kanamycin transposon THE TRANSPOSON Tn903 carrying a gene for kanamycin resistance, is 3,100 base pairs in size and contains an inverted repeat The Tn903 fragment contained in pUC4K extends from bases 1052 to 2264 instead of bases 831 to 2 264 as originally suggested (2), and the deletion appears to have occured during the initial construction of p UC4K, perhaps by tailing at a nick at base 1052. denticolacolonies. GenBank File: Plasmid sequence and annotations. Integration of this linear fragment into the lacZ locus would thereby confer the ability to grow on medium containing kanamycin, while still making use of the blue/white screen (note the selection medium contains kanamycin This procedure is commonly used to eliminate the Kanamycin resistance cassette from E. pii: AEM. 1093/nar/16. A T7-inducible IS621 recombinase is expressed from the plasmid bearing the target and bridge RNA. denticolaATCC 35405. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Hatched boxes represent zeta regions. . The approach was tested by constructing glu … a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. Asterisks indicate restriction sites present in the cassette itself but downstream of the Ana marker. coli) or ble (conferring resistance to phleomycin in both yeast and E. We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. Neal Stewart Jr recently showed that an Arabidopsis kanamycin resistance gene encodes an ATP binding cassette (ABC) transporter. An addit … The kanamycin resistance cassette was correctly positioned within the majority of genes (3,718 genes, gray). The restriction sites can be introduced by adding the corresponding sequence in the PCR primer. browse, and share richly annotated sequence files. r) allows stably transfected eukaryotic cells to be selected using G418. Com- to confer resistance to G418. Unfortunately, the reporter vector pBS3Clux also provides chloramphenicol resistance, which prevents combining these two regularly used vectors in one strain. Compared to the widely used ermF-ermAM cassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistant T. 9-kbp Neo cassette sequence containing the promoter, Neo, polyA, loxP, and Frt sites was identified in Shank3B KO mice (Figure 3A, upper panel). Feb 24, 2019 · TOPO TA 2. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. Normally, placing the drug-resistance cassette in the same orientation as the original gene works well. 015. Using the provided PCR template one can easily create a PGK-gb2-neo cassette flanked by any restriction sites to clone the cassette into the vector of choice. 2 . This confirms the suitability of the fliK locus for integration of the synthetic DNA across variable E. Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. In comparison to a wild-type version of kanaR, UTE-kanaR produced up to 30-fold more transformants in E. Furthermore, the functional reintegration of lacZ is indicated by the formation of blue colonies on X-Gal plates (e. A series of diverse ColE-like kanamycin resistance plasmids (“KanR plasmids”) from Salmonella enterica were characterized previously. If a mutant already existed such as a YKO collection mutant, genomic DNA (gDNA) was extracted from relevant YKO collection mutants and PCR was used to amplify the KO Kanamycin resistance (KanMX originating from pFA6-KanMX4) cassette with 400 base pairs up and downstream the open reading frame (ORF) using 5′/3′ UTR check primers designed Apr 15, 2005 · A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. First, … Dec 7, 1989 · We demonstrate how a kanamycin-resistance (KmR) cassette flanked by polylinkers with multiple restriction sites can be used to introduce nucleotide (nt) sequence replacements into a region of interest. pOPC-001 was obtained by combining the kanamycin resistance cassette and the I-SceI restriction site from pWRG717 , the origin of replication (R6Kγ) and origin of transfer (oriT) from pGP704 [6, 38] and the tetR and I-sceI locus from pWRG730 using Gibson Apr 17, 2015 · Sequence analysis of these two kanamycin resistance cassettes revealed that they share 35% identity and 52% similarity at the amino acid sequence level; however, only the aphA2 cassette was able to confer resistance to kanamycin in T. One limitation of the kanMX cassettes is that they are not counterselectable and therefore not readily recyclable, which is important when constructing strains Feb 4, 2008 · The correct and unique insertion of the kanamycin resistance cassette was verified by Southern. ARC sequences were defined as the sequence including an identical AR gene with identical flanking sequence, allowing for up to five base pair mismatches, in multiple unrelated isolates. GeneX represents the targeted gene. denticola ATCC 35405. Dec 1, 1995 · (nt 1-1489 in GenBank sequence V01547L The Tca cassette was originally a Pstl-EcoRl fragment (nt 1052-3175 in GenBank sequence M29725) cloned in pBR322 (where it was immediately followed by the Strategies employed for cloning a kanamycin resistance cassette Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. 1984 Gene 32: 481-485) and from a polylinker region of plasmid pJRD184 (Heusterspreute et al. RHB. necator H16 with a > 3000-fold higher efficiency (up to 10 Name Vector Type Resistance Marker Bacterial Resistance Source Sequence Available; 6xHis GFP: Unspecified: Clontech: Yes: 7. Cloning and expression of a chloramphenicol resistance gene from Campylobacter coli in 1990 [4] was followed by development of replicative cloning vectors and mutational constructs marked with chloramphenicol resistance in 1993 [5] . 1007/978-1-4939-9520-2_5. 264 (go to sequence) Sequence similarities. Arrows show directions of transcription for the expression cassette and kanamycin resistance gene. Tip. coli K-12 strain BW25113 []. These plasmids are propagated by routine methods and show similar expression profiles compared to Plasmid containing the p15A origin of replication and a kanamycin resistance gene. coli K12 strain BW25113, where each mutant contains a kanamycin resistance cassette in place of a single nonessential gene. Stephan Gruber's lab contains the insert Kanamycin resistance cassette for Bacillus subtilis selection and is published in Methods Mol Biol. Jul 1, 2006 · The high levels of resistance provided by overexpression of Atwbc19 in tobacco (Nicotiana tabacum) approached those obtained by forced expression of the nptII gene. coli. Explore the Scientific R&D Platform Scientific intelligence platform for AI-powered data management and workflow automation The appropriate location and orientation of the kanamycin resistance gene (kmr) cloned on multicopy plasmids were determined by subcloning experiments. Oct 4, 2017 · Sequence of universal KnR-cassette template, camR promoter. The pMyC backbone was amplified using the primers : 5’-agcggacctctattcacagggt and 5’-cgccccctctagctgatcac and the kanamycin resistance cassette from pMV306 was amplified using the primers 5 The kanamycin resistant pUC vectors pHSG298 and pHSG299 contain the kanamycin resistance gene, pHSG298 has the same general sequence as pHSG299 with the following The Keio single gene knockout collection comprises approximately 4000 mutants of E. UniProtKB unreviewed (TrEMBL) Organism. However, often times, the plasmid backbone includes an antibiotic resistance cassette and you aren’t introducing an additional antibiotic resistance cassette along with your gene of interest. The kanamycin resistance gene is effective at kanamycin concentrations as high as 500 µg/ml in tobacco. SnapGene File: Plasmid sequence and SnapGene enhanced annotations. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Sequence analysis of these two kana- Feb 1, 1992 · A kanamycin-resistance (KmR) cassette was modified to allow its introduction as a blunt-ended DNA fragment into target plasmids. 5. : Three kanamycin resistance gene cassettes with different polylinkers. Finally, the temperature-sensitive plasmid pCP20 was cured from these cells by growing them at 42°. Full size image The XbaI-EcoRI fragment containing the RBS sequence and the OmpA-rEPA fusion, isolated from this pM1816 vector was cloned into both pSP2 and pSP301 between the same unique restriction sites. baumannii taken from our collection were kanamycin resistant: data not shown), use of another antibiotic resistance marker such as rifampicin (for which a low level of resistance has been demonstrated in approximately 50% of multidrug Feb 15, 2022 · The right TALEN expression plasmids, containing a kanamycin resistance marker, were constructed upon inserting an expression cassette for the kanamycin resistance gene and removing the ampicillin delgarno sequence are strong enough that the . 3-kb kan cassette exhibits polarity effects in both directions. Feb 1, 1992 · A kanamycin-resistance (Km R) cassette was modified to allow its introduction as a blunt-ended DNA fragment into target plasmids. 7. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. Ken Haynes's lab contains the inserts nptII gene and 2 micron origin or replication and URA3 gene for S. 1985 Gene 37: 299-304). Kanamycin resistance in plants: an unexpected trait controlled by a potentially multifaceted gene. denticola. primers makes a smaller cassette but it is unregulated. 2015. To make selection of transgenic plants easier and more unambiguous, we replaced the gentamycin resistance cassette with either neomycin phosphotransferase II (derived from pBI101, Jefferson et al. cerevisiae and is published in Fungal Genet Biol. 3. pastoris transformants selected with G418. CRISPR advances genome engineering by directing endonuclease sequence specificity with a essential gene of E. 001 Epub 2011 Feb 13. 02. 1 (1988) 29-44. Taq polymerase master mix for recombinant checking. Plasmid pSW071 from Dr. Generally, these mutants were made by replacing the target gene sequence, spanning from the second to the seventh to the last codon based on the annotated ORFs at the time of the library construction, with a kanamycin resistance (kan R) cassette using GenBank File: Plasmid sequence and annotations. The gene (816 bp) is l … SnapGene File: Plasmid sequence and SnapGene enhanced annotations. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromoso … May 24, 2020 · Primers for generating pOPC-001, pOPC-003 and pFOK are reported in Supplementary Table S1. Subsequent excision of the Km R cassette with ApaI and religation of the plasmid leaves behind six base pairs (5′-GGGCCC) that result in the insertion of two amino acids in the target protein. First, … May 3, 2017 · The same restriction enzyme was used to insert a second copy of 2. The kanamycin cassette is effective for allelic Jan 1, 1990 · The transposon was constructed by ligating a 4. Ayalew Mentewab and C. kanamycin resistance cassette Insert Size (bp) 1600 Apr 17, 2015 · a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. 1981 Apr 5;147(2):217-26. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 am … Recombination resulted in completely replacing the gentamicin resistance cassette with HmR in all plasmids as verified by phenotype and sequence. coli strains from the Keio collection or produced by P1 transduction from Keio strains or the i>Deconvoluter library. In our laboratory experiments, the donor DNA sequence was a kanamycin resistance expression cassette, flanked with MEs. Only cells carrying plasmids that contain a kanamycin resistance cassette will grow. HindIII-digested genomic DNA of three independent colonies was hybridized with the 5' end of the kanamycin cassette, and only one band of 8016 bp was obtained confirming that the cassette was inserted into pqsC (Fig (Fig3B). Techn. tet. kanMX selector module conferring kanamycin resistance, for gene disruption in yeast, kanMX4 version. A. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a … Oct 7, 2020 · To improve compatibility with existing Golden Gate libraries, we changed the antibiotic resistance marker on the donor plasmid from bla (ampicillin resistance) to aph (3′)-Ia (kanamycin resistance). Removal of the out-of-frame ATG in the 5′ leader sequence of the NPTII gene by two different modifications increased both the levels of NPTII enzyme activity and the ID 50 for kanamycin in This is a template plasmids for frt-flanked kan cassette. This Atwbc19 protein is hypothesized to Thus, we thought of using the sequence of kanamycin marker found in commercial vectors such as pET28. The gene encoding aminoglycoside-3’-phosphotransferase (APH) that induces kanamycin resistance, was inserted to the ECKm cassette as a selection factor. 2019;2004:49-61. Alternatively, plasmid pKD4 for generating kanamycin cassette flanked by FRT sites can be used if the resistance cassette must be removed later (see Note 1). xll ytxd nrlcsk azmmh ohjv fge hpxte bwsdka xuqf fxqw