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pET His6-TwinStrep-SUMO-LwCas13a vector

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Cat No. Plasmid Name Status Add to cart
V10159 pET His6-TwinStrep-SUMO-LwCas13a

Basic Vector Information

LwCas13a refers to Cas13a from Leptotrichia wadei. CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. Download Sequencing Data for His6-TwinStrep-SUMO-LwCas13a(*.ab1)

Basic Vector Information
Vector Name pET His6-TwinStrep-SUMO-LwCas13a Antibiotic Resistance Ampicillin
Length 9549 bp Type pET & Duet Vectors (Novagen)
Source NovoPro Copy Number High copy number
Promoter T7 Cloning Method Enzyme Cut
5' Primer T7 promoter 3' Primer T7 terminator
Fusion Tag 6*His, Strep-Tag II, SUMO Expression Method IPTG induced

pET His6-TwinStrep-SUMO-LwCas13a vector Vector Map

pET His6-TwinStrep-SUMO-LwCas13a vector Vector Map

pET His6-TwinStrep-SUMO-LwCas13a vector Sequence

Copy Sequence

Download SnapGene Map(.dna)

Download GeneBank File(.gb)

LOCUS       Exported                9549 bp ds-DNA    circular SYN 15-5-2017
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    pET-His6-SUMO-LwCas13a
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9549)
  AUTHORS   xsan
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9549
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        104..208
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             209..1069
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2014..2156
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3761..4843)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4844..4921)
                     /gene="lacI"
                     /note="lacI promoter"
                     /note="
                     "
     promoter        5230..5248
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5249..5273
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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)."
     CDS             5329..5346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             5356..5373
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             5386..5409
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /note="Strep-Tag II"
                     /translation="WSHPQFEK"
     CDS             5446..5469
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /note="Strep-Tag II"
                     /translation="WSHPQFEK"
     CDS             5470..5761
                     /codon_start=1
                     /note="SUMO"
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     CDS             5767..9222
                     /codon_start=1
                     /note="LwCas13a"
                     /translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY
                     IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY
                     DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN
                     NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK
                     YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD
                     KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL
                     NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE
                     NENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE
                     IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
                     IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
                     SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
                     KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
                     SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
                     TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
                     ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
                     IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
                     GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
                     IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY
                     IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
                     IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
     terminator      9290..9337
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(9512..9540)
                     /note="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"