Basic Vector Information
- Vector Name:
- SQT1665
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8653 bp
- Type:
- Mammalian Expression, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- chicken β-actin
- 5' Primer:
- pCAG-F (5'-GCAACGTGCTGGTTATTGTG-3')
- 3' Primer:
- M13-rev
SQT1665 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 ℃.
SQT1665 vector Sequence
LOCUS 40924_48838 8653 bp DNA circular SYN 13-MAY-2021 DEFINITION Human expression plasmid for LbCpf1-NLS-3xHA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8653) AUTHORS Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK TITLE Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. JOURNAL Nat Biotechnol. 2016 Jun 27. doi: 10.1038/nbt.3620. PUBMED 27347757 REFERENCE 2 (bases 1 to 8653) TITLE Direct Submission REFERENCE 3 (bases 1 to 8653) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Biotechnol. 2016 Jun 27. doi: 10.1038/nbt.3620." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..8653 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 4..383 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 386..661 /label=chicken beta-actin promoter intron 662..1670 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1678..1697 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 1769..5452 /codon_start=1 /label=LbCpf1 /note="CRISPR-associated protein from Lachnospiraceae bacterium ND2006 (Zetsche et al., 2015)" /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS VKH" CDS 5453..5500 /codon_start=1 /label=nucleoplasmin NLS /note="bipartite nuclear localization signal from nucleoplasmin" /translation="KRPAATKKAGQAKKKK" CDS 5507..5587 /codon_start=1 /label=3xHA /note="three tandem HA epitope tags" /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" primer_bind complement(5677..5696) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 5742..5797 /label=beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(5796..5815) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(6156..6172) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(6180..6196) /label=lac operator /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(6204..6234) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(6249..6270) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 6328..6524 /label=SV40 promoter /note="SV40 early promoter" polyA_signal 6530..6664 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6732..6749) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6903..7491) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7665..8522) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8523..8627) /label=AmpR promoter
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