Basic Vector Information
- Vector Name:
- AAVS1-Puro cTnT FUCCI
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11876 bp
- Type:
- Gene knockin
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Puro
- Promoter:
- CMV
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
AAVS1-Puro cTnT FUCCI 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 ℃.
AAVS1-Puro cTnT FUCCI vector Sequence
LOCUS 62056_251 11876 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11876)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11876
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(1..837)
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
protein_bind 911..932
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 947..977
/label=lac promoter
/note="promoter for the E. coli lac operon"
polyA_signal complement(1386..1441)
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
misc_feature complement(2124..2712)
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(2750..3022)
/codon_start=1
/label=Cdt1 (30-120)
/note="degron consisting of residues 30-120 of human Cdt1
(Zielke and Edgar, 2015)"
/translation="PSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSV
DEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI"
CDS complement(3077..3727)
/codon_start=1
/label=mKO2
/note="monomeric Kusabira-Orange 2 fluorescent protein"
/translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
misc_feature complement(3734..4314)
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS complement(4679..5395)
/codon_start=1
/label=Clover
/note="bright green-yellow fluorescent protein derived from
GFP (Lam et al., 2012)"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
EFVTAAGITHGMDELYK"
protein_bind complement(5411..5435)
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
promoter complement(5467..5485)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal complement(6129..6353)
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
CDS complement(6394..6990)
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS complement(7000..7053)
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
misc_feature complement(7077..7102)
/label=SA
/note="splice acceptor site"
misc_feature complement(7109..7912)
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
promoter complement(7951..7969)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter complement(8038..8068)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(8083..8104)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(8392..8980)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 9104..9961
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
CDS complement(11364..11663)
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 11801..11817
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 11824..11842
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
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