Human EEA1 ORF clone (NM_003566) (Cat. No.: V034219)
Note: 1 NT, 1 AA Mismatches
- Name:
- pCMV-Neo-EEA1(human)-mScarlet
- Accession ID:
- NM_003566.4
- Antibiotic Resistance:
- Kanamycin
- Length:
- 8907 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Neo/G418;mScarlet
- Promoter:
- CMV
- 3' Primer:
- Sv40-polyA-R
- Fusion Tag:
- mScarlet
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
- Expression Method:
- Transient
- Gene Synonyms:
- MST105; MSTP105; ZFYVE2
- Transcript Definition:
- Homo sapiens early endosome antigen 1 (EEA1), mRNA
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
Human EEA1 ORF clone (NM_003566) (Cat. No.: V034219) Sequence
LOCUS V034219 8907 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V034219
VERSION V034219
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
enhancer 98..401
/note="human cytomegalovirus immediate early enhancer"
/label="CMV enhancer"
promoter 402..605
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 650..1345
/codon_start=1
/product="bright monomeric red fluorescent protein evolved
from a synthetic template (Bindels et al., 2016)"
/transl_table=1
/translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
STGGMDELYK"
/label="mScarlet"
CDS 1367..5602
/codon_start=1
/product="human early endosome antigen 1"
/transl_table=1
/translation="MLRRILQRTPGRVGSQGSDLDSSATPINTVDVNNESSSEGFICPQ
CMKSLGSADELFKHYEAVHDAGNDSGHGGESNLALKRDDVTLLRQEVQDLQASLKEEKW
YSEELKKELEKYQGLQQQEAKPDGLVTDSSAELQSLEQQLEEAQTENFNIKQMKDLFEQ
KAAQLATEIADIKSKYDEERSLREAAEQKVTRLTEELNKEATVIQDLKTELLQRPGIED
VAVLKKELVQVQTLMDNMTLERERESEKLKDECKKLQSQYASSEATISQLRSELAKGPQ
EVAVYVQELQKLKSSVNELTQKNQTLTENLLKKEQDYTKLEEKHNEESVSKKNIQATLH
QKDLDCQQLQSRLSASETSLHRIHVELSEKGEATQKLKEELSEVETKYQHLKAEFKQLQ
QQREEKEQHGLQLQSEINQLHSKLLETERQLGEAHGRLKEQRQLSSEKLMDKEQQVADL
QLKLSRLEEQLKEKVTNSTELQHQLDKTKQQHQEQQALQQSTTAKLREAQNDLEQVLRQ
IGDKDQKIQNLEALLQKSKENISLLEKEREDLYAKIQAGEGETAVLNQLQEKNHTLQEQ
VTQLTEKLKNQSESHKQAQENLHDQVQEQKAHLRAAQDRVLSLETSVNELNSQLNESKE
KVSQLDIQIKAKTELLLSAEAAKTAQRADLQNHLDTAQNALQDKQQELNKITTQLDQVT
AKLQDKQEHCSQLESHLKEYKEKYLSLEQKTEELEGQIKKLEADSLEVKASKEQALQDL
QQQRQLNTDLELRATELSKQLEMEKEIVSSTRLDLQKKSEALESIKQKLTKQEEEKQIL
KQDFETLSQETKIQHEELNNRIQTTVTELQKVKMEKEALMTELSTVKDKLSKVSDSLKN
SKSEFEKENQKGKAAILDLEKTCKELKHQLQVQMENTLKEQKELKKSLEKEKEASHQLK
LELNSMQEQLIQAQNTLKQNEKEEQQLQGNINELKQSSEQKKKQIEALQGELKIAVLQK
TELENKLQQQLTQAAQELAAEKEKISVLQNNYEKSQETFKQLQSDFYGRESELLATRQD
LKSVEEKLSLAQEDLISNRNQIGNQNKLIQELKTAKATLEQDSAKKEQQLQERCKALQD
IQKEKSLKEKELVNEKSKLAEIEEIKCRQEKEITKLNEELKSHKLESIKEITNLKDAKQ
LLIQQKLELQGKADSLKAAVEQEKRNQQILKDQVKKEEEELKKEFIEKEAKLHSEIKEK
EVGMKKHEENEAKLTMQITALNENLGTVKKEWQSSQRRVSELEKQTDDLRGEIAVLEAT
VQNNQDERRALLERCLKGEGEIEKLQTKVLELQRKLDNTTAAVQELGRENQSLQIKHTQ
ALNRKWAEDNEVQNCMACGKGFSVTVRRHHCRQCGNIFCAECSAKNALTPSSKKPVRVC
DACFNDLQG*"
/label="EEA1"
polyA_signal 5732..5853
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin complement(5860..6315)
/direction=LEFT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
promoter 6342..6446
/gene="bla"
/label="AmpR promoter"
promoter 6448..6805
/note="SV40 enhancer and early promoter"
/label="SV40 promoter"
CDS 6840..7634
/codon_start=1
/gene="aph(3')-II (or nptII)"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin®)"
/product="aminoglycoside phosphotransferase from Tn5"
/transl_table=1
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
/label="NeoR/KanR"
polyA_signal 7866..7913
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
/label="HSV TK poly(A) signal"
rep_origin 8242..8830
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"