Basic Vector Information
GAPTrapT2AIresMeo vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
GAPTrapT2AIresMeo vector Sequence
LOCUS Exported 12005 bp ds-DNA circular SYN 17-DEC-2018
DEFINITION Cloning vector GAPTrapT2AIresMeo, complete sequence.
ACCESSION KX783039
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12005)
AUTHORS Kao T, Labonne T, Niclis JC, Chaurasia R, Lokmic Z, Qian E, Bruveris
FF, Howden SE, Motazedian A, Schiesser JV, Costa M, Sourris K, Ng E,
Anderson D, Giudice A, Farlie P, Cheung M, Lamande SR, Penington AJ,
Parish CL, Thomson LH, Rafii A, Elliott DA, Elefanty AG, Stanley EG.
TITLE GAPTrap: A Simple Expression System for Pluripotent Stem Cells and
Their Derivatives
JOURNAL Stem Cell Reports (2016) In press
PUBMED 27594589
REFERENCE 2 (bases 1 to 12005)
AUTHORS Stanley EG, Elefanty AG, Kao T, Labonne T.
TITLE Direct Submission
JOURNAL Submitted (25-AUG-2016) Cell Biology, Murdoch Childrens Research
Institute, Royal Children's Hospital, Flemington Road, Melbourne,
Victoria 3052, Australia
REFERENCE 3 (bases 1 to 12005)
AUTHORS .
TITLE Direct Submission
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..12005
/organism="Cloning vector GAPTrapT2AIresMeo"
/mol_type="other DNA"
/db_xref="taxon:1899385"
gene 24..989
/gene="ampR"
/label=ampR
/note="codes ampicillin resistance"
regulatory 24..128
/regulatory_class="promoter"
/gene="ampR"
promoter 24..128
/gene="bla"
/label=AmpR promoter
CDS 129..989
/codon_start=1
/gene="ampR"
/product="AmpR"
/label=ampR
/protein_id="AOO95911.1"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1160..11748
rep_origin 1160..1748
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2036..2057
/label=CAP binding site
/bound_moiety="CAP"
regulatory 2072..2102
/regulatory_class="promoter"
/note="lac promoter"
promoter 2072..2102
/label=lac promoter
/note="promoter for the E. coli lac operon"
misc_feature 2110..2126
/label=lac operator
/note="lac operator"
protein_bind 2110..2126
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 2134..2150
/label=M13 rev
/note="M13 rev"
misc_feature 2146..2200
/label=lacZ-alpha
/note="lacZ-alpha"
regulatory 2171..2189
/regulatory_class="promoter"
/note="T3 promoter"
promoter 2171..2189
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
gene 2216..5559
/gene="GAPDH"
/label=GAPDH
/note="glyceraldehyde-3-phosphate dehydrogenase"
misc_feature 2219..5556
/gene="GAPDH"
/label=left arm
/note="left arm"
misc_feature 5560..5658
/label=T2A
/note="T2A"
CDS 5584..5637
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
misc_feature 5660..6246
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
regulatory 5684..6246
/regulatory_class="attenuator"
/note="IRES"
CDS 6247..7059
/codon_start=1
/product="Meo"
/label=Meo
/note="codes for G418 resistance"
/protein_id="AOO95910.1"
/translation="MARMARGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRL
SAQGRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVP
GQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDD
LDEEHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVAD
RYQDIALATRDIAEELCGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
misc_feature 7089..11324
/label=right arm
/note="right arm"
misc_feature 11348..12005
/label=pBluescript II KS(-)
/note="pBluescript II KS(-)"
promoter complement(11362..11380)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(11390..11406)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin complement(11547..12002)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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