Basic Vector Information
- Vector Name:
- pEM-Cas9HF1-recA56
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 7953 bp
- Type:
- Bacterial Expression
- Replication origin:
- p15A ori
- Copy Number:
- Low Copy
- Promoter:
- proD
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- gctcccgctgcttttaaatat
pEM-Cas9HF1-recA56 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 ℃.
pEM-Cas9HF1-recA56 vector Sequence
LOCUS 40924_17349 7953 bp DNA circular SYN 13-MAY-2021 DEFINITION Modified from pEM-Cas9HF1 (Addgene ID: 89961) to include constitutive recA56 to block recA-mediated double-strand break repair.. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7953) AUTHORS Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar R, Lynch MD TITLE Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity. JOURNAL ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174. PUBMED 28915012 REFERENCE 2 (bases 1 to 7953) TITLE Direct Submission REFERENCE 3 (bases 1 to 7953) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..7953 /mol_type="other DNA" /organism="synthetic DNA construct" CDS complement(13..636) /codon_start=1 /label=TetR /note="tetracycline repressor TetR" /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" promoter 684..739 /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" CDS 775..4878 /codon_start=1 /label=SpCas9-HF1 /note="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Kleinstiver et al., 2016)" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4879..4911 /codon_start=1 /label=ssrA tag /note="C-terminal peptide that mediates degradation in bacteria through the ClpXP and ClpAP proteases (McGinness et al., 2006)" /translation="AANDENYALAA" CDS 5030..6088 /codon_start=1 /label=RecA /note="DNA repair protein from E. coli" /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS LSLDIALGAGGLPMGCIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV DDSEGVAETNEDF" terminator 6121..6192 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6208..6235 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(6263..6280) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6397..6942) /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." terminator complement(7056..7150) /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(7174..7830) /codon_start=1 /label=CmR /note="chloramphenicol acetyltransferase" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" promoter complement(7831..7933) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase"
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