Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | AAVS1-Blasticidin-CAG-Flpe-ERT2 | Antibiotic Resistance | Ampicillin |
| Length | 12382 bp | Type | Mammalian Expression, Synthetic Biology |
| Selection Marker | Blasticidin | Copy Number | High Copy |
| Promoter | CAG | Cloning Method | Restriction Enzyme |
| 5' Primer | ggcttctggcgtgtgaccggc | 3' Primer | catagcgtaaaaggagcaaca |
AAVS1-Blasticidin-CAG-Flpe-ERT2 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-Blasticidin-CAG-Flpe-ERT2 vector Sequence
LOCUS Exported 12382 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION AAVS1 targeting donor plasmid with Flpe-ERT2 expression cassette and
blasticidin selection gene.
ACCESSION .
VERSION .
KEYWORDS AAVS1-Blasticidin-CAG-Flpe-ERT2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12382)
AUTHORS Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z,
Zhang SC
TITLE Engineering Human Stem Cell Lines with Inducible Gene Knockout using
CRISPR/Cas9.
JOURNAL Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi:
10.1016/j.stem.2015.06.001.
PUBMED 26145478
REFERENCE 2 (bases 1 to 12382)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell Stem
Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi:
10.1016/j.stem.2015.06.001."
FEATURES Location/Qualifiers
source 1..12382
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 33..836
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
misc_feature 843..868
/label=SA
/note="splice acceptor site"
CDS 892..945
/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"
primer_bind complement(1418..1435)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1424..1648
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
enhancer 1716..2095
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2097..2374
/label=chicken beta-actin promoter
intron 2376..3393
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 3317..3339
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 3401..3420
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 3457..3466
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4762..5697
/codon_start=1
/product="mutated ligand-binding domain of the human
estrogen receptor (Feil et al., 1997)"
/label=ERT2
/translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
misc_feature 5737..6325
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5790..5810)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6208..6219)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 6357..6833
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(6556..6575)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
primer_bind complement(6943..6962)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 7008..7063
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(7062..7081)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(7424..7440)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 7448..7464
/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)."
promoter complement(7472..7502)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7517..7538
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
misc_feature 7612..8448
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
primer_bind complement(8488..8507)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(8489..8507)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(8514..8531)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(8514..8530)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8523..8545)
/label=M13/pUC Forward
/note="In lacZ gene"
CDS 8668..8970
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 8849..8868
/label=ccdB-fwd
/note="ccdB gene, forward primer"
primer_bind complement(9373..9392)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 9987..10006
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
CDS complement(10367..11227)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 10990..11009
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 11351..11939
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11840..11859
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 12093..12110
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 12227..12248
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 12263..12293
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12301..12317
/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 12325..12341
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 12362..12380
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
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