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    New England Biolabs i scei
    Schematic of the gene cluster assembly in the yTREX vector. A. The yTREX vector backbone comprises replication elements and selection markers for E. coli (ori, pMB 1 origin of replication; Km R , kanamycin resistance gene) and yeast ( CEN 4 / ARS 1 , S. cerevisiae centromere region and autonomously replicating sequence; URA 3 , orotidine 5′‐phosphate decarboxylase gene) and the yTREX cassettes. L‐ yTREX (orange): oriT, origin of transfer; OE , outside end of transposon Tn5; P T 7 , T7 bacteriophage promoter, R‐ yTREX (green): tnp , Tn5 transposase gene; OE ; Tc R , tetracycline resistance gene; P T 7 . The vector is linearized by hydrolysis with restriction endonuclease I‐ <t>Sce</t> I, thereby exposing the partial I‐ <t>Sce</t> I recognition site and the sequences of the CIS (cluster integration site) at the termini. At the respective CIS 1 and CIS 2 sequences, insert fragments with appropriate homology arms to the CIS sequences and to one another can be integrated via yeast recombineering. Depiction is not drawn to scale. The complete vector sequence is available at the NCBI database (GenBank MK416190) and in the Table S1 in GenBank format. Right panel: Creation of homologous regions for recombination can generally be achieved by PCR and appropriate positioning of fully binding primers. Accordingly, designed primers can be used to re‐assemble large gene clusters in their native organization from freely defined PCR fragments (B). Alternatively, the use of primers with 5′‐elongations adding sequences to match new adjacent fragments enables re‐arrangements of genes or the addition of new parts (C). In this case, primer positions are defined by the ends of the fragments that are to be connected. Find further information under section Generation of gene cluster DNA fragments yeast assembly cloning in the yTREX vector , step 3b.
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    Schematic of the gene cluster assembly in the yTREX vector. A. The yTREX vector backbone comprises replication elements and selection markers for E. coli (ori, pMB 1 origin of replication; Km R , kanamycin resistance gene) and yeast ( CEN 4 / ARS 1 , S. cerevisiae centromere region and autonomously replicating sequence; URA 3 , orotidine 5′‐phosphate decarboxylase gene) and the yTREX cassettes. L‐ yTREX (orange): oriT, origin of transfer; OE , outside end of transposon Tn5; P T 7 , T7 bacteriophage promoter, R‐ yTREX (green): tnp , Tn5 transposase gene; OE ; Tc R , tetracycline resistance gene; P T 7 . The vector is linearized by hydrolysis with restriction endonuclease I‐ Sce I, thereby exposing the partial I‐ Sce I recognition site and the sequences of the CIS (cluster integration site) at the termini. At the respective CIS 1 and CIS 2 sequences, insert fragments with appropriate homology arms to the CIS sequences and to one another can be integrated via yeast recombineering. Depiction is not drawn to scale. The complete vector sequence is available at the NCBI database (GenBank MK416190) and in the Table S1 in GenBank format. Right panel: Creation of homologous regions for recombination can generally be achieved by PCR and appropriate positioning of fully binding primers. Accordingly, designed primers can be used to re‐assemble large gene clusters in their native organization from freely defined PCR fragments (B). Alternatively, the use of primers with 5′‐elongations adding sequences to match new adjacent fragments enables re‐arrangements of genes or the addition of new parts (C). In this case, primer positions are defined by the ends of the fragments that are to be connected. Find further information under section Generation of gene cluster DNA fragments yeast assembly cloning in the yTREX vector , step 3b.

    Journal: Microbial Biotechnology

    Article Title: Protocols for yTREX/Tn5‐based gene cluster expression in Pseudomonas putida

    doi: 10.1111/1751-7915.13402

    Figure Lengend Snippet: Schematic of the gene cluster assembly in the yTREX vector. A. The yTREX vector backbone comprises replication elements and selection markers for E. coli (ori, pMB 1 origin of replication; Km R , kanamycin resistance gene) and yeast ( CEN 4 / ARS 1 , S. cerevisiae centromere region and autonomously replicating sequence; URA 3 , orotidine 5′‐phosphate decarboxylase gene) and the yTREX cassettes. L‐ yTREX (orange): oriT, origin of transfer; OE , outside end of transposon Tn5; P T 7 , T7 bacteriophage promoter, R‐ yTREX (green): tnp , Tn5 transposase gene; OE ; Tc R , tetracycline resistance gene; P T 7 . The vector is linearized by hydrolysis with restriction endonuclease I‐ Sce I, thereby exposing the partial I‐ Sce I recognition site and the sequences of the CIS (cluster integration site) at the termini. At the respective CIS 1 and CIS 2 sequences, insert fragments with appropriate homology arms to the CIS sequences and to one another can be integrated via yeast recombineering. Depiction is not drawn to scale. The complete vector sequence is available at the NCBI database (GenBank MK416190) and in the Table S1 in GenBank format. Right panel: Creation of homologous regions for recombination can generally be achieved by PCR and appropriate positioning of fully binding primers. Accordingly, designed primers can be used to re‐assemble large gene clusters in their native organization from freely defined PCR fragments (B). Alternatively, the use of primers with 5′‐elongations adding sequences to match new adjacent fragments enables re‐arrangements of genes or the addition of new parts (C). In this case, primer positions are defined by the ends of the fragments that are to be connected. Find further information under section Generation of gene cluster DNA fragments yeast assembly cloning in the yTREX vector , step 3b.

    Article Snippet: Step 1: To linearize the yTREX vector by hydrolyzation using I‐Sce I restriction endonuclease, prepare a 30 μl reaction mixture with 1 μl I‐Sce I (5 U), the manufacturer's buffer (New England Biolabs GmbH, Ipswich, USA) as well as ˜2 μg vector DNA, and incubate for 16 h at 37°C (according to the manufacturer's specifications, a lower amount of enzyme (1–2 U) would also be sufficient for this step).

    Techniques: Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Binding Assay, Clone Assay

    Comparison between different gene targeting strategies. Ends-out GT steps are generation of linear donor DNA, homologous recombination between donor DNA and targeted sequences, and recovery of correct GT. (A) For the Golic heat-shock strategy, the donor is first inserted in the genome as a P -element transgene and then released (flip-out and linearization) in larval primordial germ cells by heat-shock-induced expression of FLP and I- Sce I. Targeting occurs rarely through endogenous homologous recombination machinery. Candidates, possibly carrying the same GT event due to later clonal expansion, are recovered based on the mini-white eye marker in between the 5′ and 3′ homology arms. (B) For embryo microinjection, donor DNA is injected together with the corresponding sequence-specific nuclease to boost GT in embryonic pole cells. Clonal expansion can again lead to multiple offspring carrying identical GT. (C) In Golic+, donor DNA is not released from the transgene until the birth of each cystoblast (CB) from the ovarian germline stem cells, guaranteeing independent GT among candidates. Ends-out GT in CBs requires DNA double-strand breaks made by CRISPR/Cas, and recovery of correct GT is facilitated by a repressor-based lethality selection. The CB-specific induction of FLP, I- Sce I, and Cas9 depends on bamP-GAL4 ; guide RNA for CRISPR/Cas is broadly expressed with the dU6 promoter; strong candidates are recovered based on inheritance of a repressor, miRNA against rCD2, to rescue the pupal lethality caused by nSyb-driven riTS-Rac1 V12 .

    Journal: Genetics

    Article Title: An Enhanced Gene Targeting Toolkit for Drosophila: Golic+

    doi: 10.1534/genetics.114.173716

    Figure Lengend Snippet: Comparison between different gene targeting strategies. Ends-out GT steps are generation of linear donor DNA, homologous recombination between donor DNA and targeted sequences, and recovery of correct GT. (A) For the Golic heat-shock strategy, the donor is first inserted in the genome as a P -element transgene and then released (flip-out and linearization) in larval primordial germ cells by heat-shock-induced expression of FLP and I- Sce I. Targeting occurs rarely through endogenous homologous recombination machinery. Candidates, possibly carrying the same GT event due to later clonal expansion, are recovered based on the mini-white eye marker in between the 5′ and 3′ homology arms. (B) For embryo microinjection, donor DNA is injected together with the corresponding sequence-specific nuclease to boost GT in embryonic pole cells. Clonal expansion can again lead to multiple offspring carrying identical GT. (C) In Golic+, donor DNA is not released from the transgene until the birth of each cystoblast (CB) from the ovarian germline stem cells, guaranteeing independent GT among candidates. Ends-out GT in CBs requires DNA double-strand breaks made by CRISPR/Cas, and recovery of correct GT is facilitated by a repressor-based lethality selection. The CB-specific induction of FLP, I- Sce I, and Cas9 depends on bamP-GAL4 ; guide RNA for CRISPR/Cas is broadly expressed with the dU6 promoter; strong candidates are recovered based on inheritance of a repressor, miRNA against rCD2, to rescue the pupal lethality caused by nSyb-driven riTS-Rac1 V12 .

    Article Snippet: To create pTL2, we first removed the I- Sce I site downstream of the 3′ multiple-cloning site (MCS) of pTL1. pTL1 was partially digested with I- Sce I and recircularized using Gibson assembly [New England BioLabs (Beverly, MA), Gibson Assembly Master Mix, E2611L] with a small PCR fragment containing one Avr II site at each end.

    Techniques: Homologous Recombination, Expressing, Marker, Injection, Sequencing, CRISPR, Selection