List of entry and destination vectors

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Here is a list of the constructs in the Tol2kit. Click on any construct name (second column) to see more information. 12/18/07: Clone pages now include annotated sequence in Genbank format.

Here are insert sequences for entry clones, and 5' and 3' ends for destination clones, along with a description of how to assemble predicted sequences for expression clones.

Constructs in the Tol2kit v1.2 (Nov 2007)

# name insert size (bp) made by ver MTA?
5' entry clones, attL4-R1 (kan resistant)
299p5E-bactin2 5.3 kb beta-actin promoter (ubiquitous)7950KMK1.0
380p5E-h2afx 1 kb histone2A-X promoter (quasi-ubiquitous)3604KMK1.0
382p5E-CMV/SP6 1 kb CMV/SP6 cassette from pCS2+3704KMK1.0
222p5E-hsp70l 1.5 kb hsp70l promoter for heat-shock induction4163BM1.0
327p5E-UAS 10x UAS element and basal promoter for Gal4 response3127DSC1.0
228p5E-MCS multiple-cloning site from pBluescript2810BM1.0
381p5E-Fse-Asc restriction sites for 8-cutters FseI and AscI2663EF1.0
middle entry clones, attL1-L2 (kan resistant)
383pME-EGFP EGFP3327KMK1.0
384pME-EGFPCAAX membrane-localized (prenylated) EGFP; fused to 21 aa of H-ras3345KMK1.0
385pME-nlsEGFP nuclear-localized EGFP3342KMK1.0
386pME-mCherry monomeric red fluorophore mCherry3261KMK1.0
232pME-mCherryCAAX H80D** prenylated mCherry (deleterious H80D mutation; superseded by 450)3321KMK1.0
233pME-nlsmCherry nuclear-localized mCherry3288KMK1.0
234pME-H2AmCherry mCherry fused to zebrafish histone H2A.F/Z3651KMK1.0
387pME-Gal4VP16 Gal4 DNA binding domain fused to VP16 transactivation domain3204EF1.0
237pME-MCS multiple-cloning site from pBluescript2765BM1.1
450pME-mCherryCAAX** prenylated mCherry (some preps contam w/ H80D, superseded by #550)3321KMK/SYC1.2
455pME-EGFP no stop EGFP, no stop (to make N-terminal fusions)3324KMK1.2
456pME-mCherry no stop mCherry, no stop (to make N-terminal fusions)3258KMK1.2
550pME-mCherryCAAX prenylated mCherry3321KMK/SYC1.2
3' entry clones, attR2-L3 (kan resistant)
302p3E-polyA SV40 late polyA signal2838KMK1.0
229p3E-MTpA 6x myc tag for C-terminal fusions, plus SV40 late polyA3151BM1.0
366p3E-EGFPpA EGFP for C-terminal fusions, plus SV40 late polyA3634MEH1.0
388p3E-mCherrypA mCherry for C-terminal fusions, plus SV40 late polyA3586MEH1.0
389p3E-IRES-EGFPpA IRES driving EGFP plus SV40 late polyA4219KMK1.0
390p3E-IRES-EGFPCAAXpA IRES driving prenylated EGFP plus SV40 late polyA4250KMK1.0
391p3E-IRES-nlsEGFPpA IRES driving nuclear EGFP plus SV40 late polyA4248KMK1.0
destination vectors, attR4-R3 (amp/chlor resistant; grow in ccdB-tolerant cells)
392pDestTol2pA* attR4-R3 gate with SV40 polyA flanked by Tol2 inverted repeats7903CG0.9KK
393pDestTol2CG* pDestTol2pA with cmlc2:egfp transgenesis marker9708EF0.9KK
394pDestTol2pA2 pDestTol2pA with ~2 kb extraneous sequence removed5883EF1.0KK
395pDestTol2CG2 pDestTol2CG2 with ~2 kb extraneous sequence removed7796EF1.0KK
donor vectors (kan/chlor resistant; grow in ccdB-tolerant cells)
218pDONR221 middle donor vector; attP1-P2 flanking chlor/ccdB cassetteInvitrogen1.1
219pDONRP4-P1R 5' donor vector; attP4-P1R flanking chlor/ccdB cassetteInvitrogen1.1
220pDONRP2R-P3 3' donor vector; attP2R-P3 flanking chlor/ccdB cassetteInvitrogen1.1
transposase clones
396pCS2FA-transposase for in vitro transcription of capped Tol2 transposase mRNA6034KMK1.0KK

Notes

  • # is the Chien lab stock number
  • made by shows who made the construct:
    • CG: Clemens Grabher, Look lab
    • BM: Ben Mangum, Chien lab
    • DSC, Doug Campbell, Chien lab
    • EF, Esther Fujimoto, Chien lab
    • KMK, Kristen Kwan, Chien lab
    • MEH, Melissa Hardy, Chien lab
    • SYC, Seok-yong Choi, Chitnis lab
  • ver shows the version of the Tol2kit in which this construct first appeared.
  • *:pDestTol2pA and pDestTol2CG have been superseded by pDestTol2pA2 and pDestTol2CG2. They are not formally part of the v1.0 release, and are no longer being distributed.
  • **:clone 232 was superseded by clone 450, which lacks the H80D mutation in mCherryCAAX. Subsequently, some preps of clone 450 turned out to still be clone 232, so we cleaned it up again, and clone 450 has been superseded by 550.
  • MTA? indicates whether an MTA is associated with each clone. "KK", Koichi Kawakami.
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