STRAIGHT-IN Dual Kit
(Kit #
1000000283
)
Depositing Lab: Kate Galloway
The STRAIGHT-IN Dual kit provides a complete set of components for the seamless installation of STRAIGHT-IN (Serine and Tyrosine Recombinase-Assisted Integration of Genes for High-Throughput Investigation) landing pads in hiPSCs and potentially other cell lines. It includes three novel landing pads, programmable TALEN vectors targeting the CLYBL locus, and DASIT (Destabilized-nanobody Antigen Selection and Identification Tool) constructs for efficient enrichment of targeted clones. The kit also contains GT- and GA-donor plasmids (either empty or engineered for constitutive CAG or doxycycline-inducible expression of genes of interest), along with IVT (in vitro transcription) templates for the recombinases used in the STRAIGHT-IN platform (eeBxb1, p53DD, and Flp-T2A-BleoR), an eeBxb1 expression plasmid, and donor plasmids expressing fluorescent proteins or transcription factors as positive controls. Together, these components enable efficient and streamlined establishment of the STRAIGHT-IN Dual system.
Note that while the kit supports the installation of up to two landing pads (GT and GA) into both CLYBL alleles using TALENs, enabling the generation of STRAIGHT-IN Dual lines, users may also choose to install only a single landing pad to generate STRAIGHT-IN lines.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
Engineering STRAIGHT-IN single and dual landing pad hiPSC lines in the male iPS11 parental line for programmable DNA integration. Blanch-Asensio, A, Gumustop, DR, Wang, NB, Peterman, EL, Ploessl, DS, Galloway, KE. Stem Cell Research. 2026, In submission. Preprint (Link opens in a new window)
Description
The STRAIGHT-IN Dual kit provides a comprehensive and integrated set of components designed to enable the efficient and seamless installation of STRAIGHT-IN landing pads in human induced pluripotent stem cells (hiPSCs) and potentially a wide range of other cell types.
The kit includes three novel landing pad constructs that are introduced into the genome using programmable TALEN vectors targeting the CLYBL genomic safe harbor locus, ensuring precise and stable integration. To support enrichment of correctly targeted cells, DASIT constructs are provided for efficient selection of edited clones. In addition, the kit offers a suite of GT- and GA- STRAIGHT-IN donor plasmids, which can be used either as empty backbones with a multicloning site for insertion of a payload of interest, or as pre-engineered constructs driving gene expression under a constitutive CAG promoter or a doxycycline-inducible all-in-one Tet-On 3G system, providing flexibility for downstream applications. To facilitate site-specific recombination within the STRAIGHT-IN platform, the kit includes IVT templates encoding the required recombinases (eeBxb1, Cre, and Flp-T2A-BleoR) along with p53DD to enhance integration efficiency and a positive control for IVT and modRNA transfection (mGreenLantern). An eeBxb1 expression plasmid is also included for recombinase delivery without the need for IVT. The kit is further complemented by STRAIGHT-IN donor plasmids expressing constitutive fluorescent proteins (iRFP670 and mGreenLantern) or inducible transcription factors (ETV2 and NKX3.1), which serve as positive controls to validate system performance.
Collectively, these components provide a streamlined, modular, and scalable workflow for establishing the STRAIGHT-IN Dual system, enabling precise genome engineering with high efficiency and reproducibility.
Note that while the kit supports the installation of up to two landing pads (GT and GA) into both CLYBL alleles using TALENs, enabling the generation of STRAIGHT-IN Dual lines, users may also choose to install only a single landing pad to generate STRAIGHT-IN lines.
Figure 1: STRAIGHT-IN Dual kit containing all components required to generate STRAIGHT-IN single and dual landing pad hiPSC lines, and to perform DNA payload integration and auxiliary sequence excision within the STRAIGHT-IN platform. The kit includes: (1) TALEN and landing pad targeting constructs; (2) DASIT IVT templates for enrichment of targeted cells; (3) empty, constitutive, and doxycycline-inducible GT- and GA-Donor plasmids; (4) fluorescent protein and inducible transcription factor constructs as positive controls; (5) IVT templates for modRNA-mediated expression of eeBxb1 and p53DD for donor integration, and Cre and Flp-T2A-BleoR for auxiliary element excision; and (6) an eeBxb1 expression plasmid for DNA-mediated donor integration. Adapted from Blanch-Asensio et al., Stem Cell Research (2026). In submission.
Kit Documentation
Find additional STRAIGHT-IN protocols and plasmids in:
STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines. Blanch-Asensio A, Grandela C, Mummery CL, Davis RP. Nat Protoc. 2024 Aug 23;21(2):429-472. doi: 10.1038/s41596-024-01039-2. PubMed (Link opens in a new window) Article (Link opens in a new window) Plasmids at Addgene
STRAIGHT-IN Dual: a platform for dual single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells. Blanch-Asensio A, Ploessl DS, Johnson BB, Cascione S, Berndsen MRM, Wang NB, Orlova VV, Alemany A, Mummery CL, Galloway KE, Davis RP. Nat Biomed Eng. 2026 Apr 30. doi: 10.1038/s41551-026-01677-9. PubMed (Link opens in a new window) Article (Link opens in a new window) Plasmids at Addgene
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
"The STRAIGHT-IN Dual Kit was a gift from Kate Galloway (Addgene kit #1000000283)."
For your Reference section:
Engineering STRAIGHT-IN single and dual landing pad hiPSC lines in the male iPS11 parental line for programmable DNA integration. Blanch-Asensio, A, Gumustop, DR, Wang, NB, Peterman, EL, Ploessl, DS, Galloway, KE. Stem Cell Research. 2026, In submission. Preprint (Link opens in a new window)
STRAIGHT-IN Dual Kit - #1000000283
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Kanamycin | |
| Ampicillin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | CLYBL_Bxb1-GT_CAG-EBFP_LP _TC |
|
| A / 2 | CLYBL_Bxb1-GA_CAG-EGFP_LP _TC |
|
| A / 3 | CLYBL_Bxb1-GA_CAG-mSc-ALFA_LP _TC |
|
| A / 4 | CLYBL_CAG-TALEN-L1 |
|
| A / 5 | CLYBL_CAG-TALEN-R1 |
|
| A / 6 | Bxb1-GT-CAG_Donor_KanR |
|
| A / 7 | Bxb1-GA-CAG_Donor_KanR |
|
| A / 8 | Bxb1-GT_AIO-TetOn_Divergent_Donor_KanR |
|
| A / 9 | Bxb1-GA_AIO-TetOn_Divergent_Donor_KanR |
|
| A / 10 | Bxb1-GA-Dox-NKX3-1_Div |
|
| A / 11 | CAG-eeBxb1_bGH |
|
| A / 12 | pIVT-desNb_EGFP-PuroR-mRuby2 |
|
| B / 1 | pIVT-desNb_EGFP-BleoR-mRuby2 |
|
| B / 2 | pIVT-wtNb_ALFA-PuroR-mRuby2 |
|
| B / 3 | Bxb1-GT-Donor_CBh_mCherry |
|
| B / 4 | Bxb1-GA-Donor_CBh_mCherry |
|
| B / 5 | Bxb1-GT-CAG-mGL |
|
| B / 6 | Bxb1-GA-CAG-iRFP670 |
|
| B / 7 | Bxb1-GT-Dox-ETV2_Div |
|
| B / 8 | pIVT-Flp-2A-BleoR |
|
| B / 9 | pIVT-iCre |
|
| B / 10 | pIVT-eeBxb1 |
|
| B / 11 | pIVT-p53DD |
|
| B / 12 | pIVT-mGL |
|