Basic Vector Information
- Vector Name:
- MSP1673
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 7309 bp
- Type:
- Bacterial Expression, CRISPR
- Replication origin:
- p15A ori
- Copy Number:
- Low Copy
- Promoter:
- T7 (x2)
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- ACYC-DuetUP1
- 3' Primer:
- oBK390 (5'-GCAAATTCGACCCGGTCGTCG-3')
MSP1673 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 ℃.
MSP1673 vector Sequence
LOCUS 40924_2099 7309 bp DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression plasmid for S. thermophilus1 Cas9. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7309) AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK TITLE Engineered CRISPR-Cas9 nucleases with altered PAM specificities. JOURNAL Nature. 2015 Jun 22. doi: 10.1038/nature14592. PUBMED 26098369 REFERENCE 2 (bases 1 to 7309) TITLE Direct Submission REFERENCE 3 (bases 1 to 7309) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 2015 Jun 22. doi: 10.1038/nature14592." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..7309 /mol_type="other DNA" /organism="synthetic DNA construct" protein_bind 3..27 /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)." RBS 42..64 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 77..3436 /codon_start=1 /label=St1Cas9 /note="Cas9 endonuclease from the Streptococcus thermophilus Type II CRISPR/Cas system (Esvelt et al., 2013)" /translation="SDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVR RTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEEL FIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQ LRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKR KYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLLN DLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDK SGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTT SSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDD EKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCL YTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDS MDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVL NALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLW KKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQ VDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKF LMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPE IKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQF EKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTM PKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQH IIKNEGDKPKLDF" CDS 3449..3469 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" promoter 3538..3556 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA 3618..3742 /label=St1 gRNA scaffold /note="guide RNA scaffold for the Streptococcus thermophilus CRISPR/Cas9 system (Esvelt et al., 2013)" terminator 3761..3809 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(4033..4689) /codon_start=1 /label=CmR /note="chloramphenicol acetyltransferase" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" promoter complement(4690..4792) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind complement(5184..5201) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5318..5862) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin." protein_bind complement(6053..6074) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(6090..7169) /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(7170..7247) /label=lacI promoter
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