pGEM-WingGFP-tan
(Plasmid #83473)

• Purpose: EGFP selectable marker for use in two-stage CRISPR-mediated allele replacement strategies in Drosophila. D. americana tan homology arms can be replaced with locus of choice.
• Depositing Lab: Patricia Wittkopp
• Publication: Lamb et al Fly (Austin). 2016 Aug 5:1-12.

Backbone

• Vector backbone: pGEM T-easy
• Backbone manufacturer: Promega
• Backbone size w/o insert (bp): 3015
• Total vector size (bp): 7290
• Vector type: Bacterial Expression, Insect Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: WingGFP-tan
• Insert Size (bp): 4275
• Promoter: Hsp70 and enhancer from D. melanogaster yellow gene for pupal wing expression

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: M13-F
• 3′ sequencing primer: M13 Reverse

Resource Information

• Supplemental Documents:
  • supplemental sequence info

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

To be used as a selectable marker in two-stage CRISPR-mediated allele replacement strategies as described in Lamb, et al 2016. This donor contains a selectable green fluorescent protein driven by an enhancer from the D. melanogaster yellow gene that can be screened in the developing pupal wing. The current insert contains homology arms targeting the D. americana tan gene, which can be replaced with homology arms targeting the experimenter's locus of choice.
Construct contains:
5’ homology arm
5’ target PAM
Unique portion of 5’ target site for reporter excision (restriction sites: BglII, BsiWI, Acc65I)
Pupal wing enhancer from D. melanogaster yellow gene
EGFP with Hsp70 promoter and SV40 polyadenylation signal
Unique portion of 3’ target site for reporter excision (restriction sites: NarI, Bsu36I, ClaI)
3’ homology arm
3’ target PAM

Restriction sites within pGEM backbone for homology arm removal: 5’ end – ApaI, SphI; 3’ end – SbfI, MluI