Human CCNT1 ORF clone (NM_001240) (Cat. No.: V026099)
Note: This is a 6895 bp mammalian expression vector, pCMV-SV40-Neo-CCNT1(human)-mCherry. It is designed for expressing the human CCNT1 gene fused with mCherry, driven by a strong CMV promoter. A NeoR/KanR gene under the SV40 promoter allows for antibiotic selection. The vector also includes an f1 ori for single-stranded DNA production and an AmpR/ori cassette for bacterial propagation.
- Name:
- pCMV-SV40-Neo-CCNT1(human)-mCherry
- Accession ID:
- NM_001240.4
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6895 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Neo/G418;mCherry
- Promoter:
- CMV
- Fusion Tag:
- mCherry
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 30℃
- Expression Method:
- Transient
- Gene Synonyms:
- CCNT; CYCT1; HIVE1
- Transcript Definition:
- Homo sapiens cyclin T1 (CCNT1), transcript variant a, 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 CCNT1 ORF clone (NM_001240) (Cat. No.: V026099) Sequence
LOCUS V026099 6895 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V026099
VERSION V026099
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
enhancer 61..364
/note="human cytomegalovirus immediate early enhancer"
/label="CMV enhancer"
promoter 365..568
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 641..2818
/label="CCNT1(NM_001240)"
/note="CCNT1(NM_001240)"
/gene="CCNT1"
CDS 2852..3562
/codon_start=1
/note="mammalian codon-optimized"
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/transl_table=1
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK*"
/label="mCherry"
polyA_signal 3683..3804
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin complement(3811..4266)
/direction=LEFT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
promoter 4293..4397
/gene="bla"
/label="AmpR promoter"
promoter 4399..4756
/note="SV40 enhancer and early promoter"
/label="SV40 promoter"
CDS 4791..5585
/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 5817..5864
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
/label="HSV TK poly(A) signal"
rep_origin 6193..6781
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"