pSL1142 (pSPIN, pBBR1 backbone) vector (V006643)

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V006643	pSL1142 (pSPIN, pBBR1 backbone)	In stock, instant shipping	Buy one, get one free!

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Single-plasmid V. cholerae CAST system, encodes all proteins, crRNA, and donor DNA. Entry vector encodes non-targeting crRNA, with BsaI sites for spacer cloning. pBBR1 backbone.

Vector Name:: pSL1142 (pSPIN, pBBR1 backbone)

Antibiotic Resistance:: Kanamycin

Length:: 14031 bp

Type:: Bacterial Expression

Replication origin:: pBBR1 oriV

Copy Number:: Low Copy

Promoter:: J23119(SpeI)

Cloning Method:: Gibson Cloning

5' Primer:: cgccttcttgacgagttc

3' Primer:: GCTAGTTATTGCTCAGCGG

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pSL1142 (pSPIN, pBBR1 backbone) vector Map

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Plasmid 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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat Biotechnol. 2021 Apr;39(4):480-489. doi: 10.1038/s41587-020-00745-y. Epub 2020 Nov 23.

pSL1142 (pSPIN, pBBR1 backbone) vector Sequence

LOCUS       Exported               14031 bp DNA     circular SYN 03-DEC-2024
DEFINITION  Single-plasmid V. cholerae INTEGRATE system, encodes all proteins, 
            crRNA, and donor DNA. Entry vector encodes non-targeting crRNA, with
            BsaI sites for spacer cloning. pBBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    pSL1142
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14031)
  AUTHORS   Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH
  TITLE     CRISPR RNA-guided integrases for high-efficiency, multiplexed 
            bacterial genome engineering.
  JOURNAL   Nat Biotechnol. 2020 Nov 23. pii: 10.1038/s41587-020-00745-y. doi: 
            10.1038/s41587-020-00745-y.
  PUBMED    33230293
REFERENCE   2  (bases 1 to 14031)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 14031)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 14031)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Nov 23. pii: 10.1038/s41587-020-00745-y. doi: 
            10.1038/s41587-020-00745-y."
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..14031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(122..893)
                     /direction=LEFT
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     misc_feature    998..1049
                     /label=oriT
     CDS             1117..2115
                     /codon_start=1
                     /product="MobV family relaxase"
                     /label=Mob
                     /translation="MAAYAIMRCKKLAKMGNVAASLKHAYRERETPNADASRTPENEHW
                     AASSTDEAMGRLRELLPEKRRKDAVLAVEYVMTASPEWWKSASQEQQAAFFEKAHKWLA
                     DKYGADRIVTASIHRDETSPHMTAFVVPLTQDGRLSAKEFIGNKAQMTRDQTTFAAAVA
                     DLGLQRGIEGSKARHTRIQAFYEALERPPVGHVTISPQAVEPRAYAPQGLAEKLGISKR
                     VETPEAVADRLTKAVRQGYEPALQAAAGAREMRKKADQAQETARDLRERLKPVLDALGP
                     LNRDMQAKAAAIIKAVGEKLLTEQREVQRQKQAQRQQERGRAHFPEKCHLG"
     CDS             2373..3164
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        3639..3673
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119)"
     CDS             3816..5000
                     /codon_start=1
                     /label=TniQ
                     /note="TniQ family protein [Vibrio cholerae]"
                     /translation="MFLQRPKPYSDESLESFFIRVANKNGYGDVHRFLEATKRFLQDID
                     HNGYQTFPTDITRINPYSAKNSSSARTASFLKLAQLTFNEPPELLGLAINRTNMKYSPS
                     TSAVVRGAEVFPRSLLRTHSIPCCPLCLRENGYASYLWHFQGYEYCHSHNVPLITTCSC
                     GKEFDYRVSGLKGICCKCKEPITLTSRENGHEAACTVSNWLAGHESKPLPNLPKSYRWG
                     LVHWWMGIKDSEFDHFSFVQFFSNWPRSFHSIIEDEVEFNLEHAVVSTSELRLKDLLGR
                     LFFGSIRLPERNLQHNIILGELLCYLENRLWQDKGLIANLKMNALEATVMLNCSLDQIA
                     SMVEQRILKPNRKSKPNSPLDVTDYLFHFGDIFCLWLAEFQSDEFNRSFYVSRW"
     CDS             5001..6923
                     /codon_start=1
                     /label=Cas8
                     /note="Cas8 from Vibrio vulnificus, type I-F 
                     CRISPR-associated protein Csy2"
                     /translation="MQTLKELIASNPDDLTTELKRAFRPLTPHIAIDGNELDALTILVN
                     LTDKTDDQKDLLDRAKCKQKLRDEKWWASCINCVNYRQSHNPKFPDIRSEGVIRTQALG
                     ELPSFLLSSSKIPPYHWSYSHDSKYVNKSAFLTNEFCWDGEISCLGELLKDADHPLWNT
                     LKKLGCSQKTCKAMAKQLADITLTTINVTLAPNYLTQISLPDSDTSYISLSPVASLSMQ
                     SHFHQRLQDENRHSAITRFSRTTNMGVTAMTCGGAFRMLKSGAKFSSPPHHRLNSKRSW
                     LTSEHVQSLKQYQRLNKSLIPENSRIALRRKYKIELQNMVRSWFAMQDHTLDSNILIQH
                     LNHDLSYLGATKRFAYDPAMTKLFTELLKRELSNSINNGEQHTNGSFLVLPNIRVCGAT
                     ALSSPVTVGIPSLTAFFGFVHAFERNINRTTSSFRVESFAICVHQLHVEKRGLTAEFVE
                     KGDGTISAPATRDDWQCDVVFSLILNTNFAQHIDQDTLVTSLPKRLARGSAKIAIDDFK
                     HINSFSTLETAIESLPIEAGRWLSLYAQSNNNLSDLLAAMTEDHQLMASCVGYHLLEEP
                     KDKPNSLRGYKHAIAECIIGLINSITFSSETDPNTIFWSLKNYQNYLVVQPRSINDETT
                     DKSSL"
     CDS             6895..7953
                     /codon_start=1
                     /label=Cas7
                     /note="type I-F CRISPR-associated protein Cas7 [Vibrio 
                     cholerae]"
                     /translation="MKLPTNLAYERSIDPSDVCFFVVWPDDRKTPLTYNSRTLLGQMEA
                     ASLAYDVSGQPIKSATAEALAQGNPHQVDFCHVPYGASHIECSFSVSFSSELRQPYKCN
                     SSKVKQTLVQLVELYETKIGWTELATRYLMNICNGKWLWKNTRKAYCWNIVLTPWPWNG
                     EKVGFEDIRTNYTSRQDFKNNKNWSAIVEMIKTAFSSTDGLAIFEVRATLHLPTNAMVR
                     PSQVFTEKESGSKSKSKTQNSRVFQSTTIDGERSPILGAFKTGAAIATIDDWYPEATEP
                     LRVGRFGVHREDVTCYRHPSTGKDFFSILQQAEHYIEVLSANKTPAQETINDMHFLMAN
                     LIKGGMFQHKGD"
     CDS             7956..8555
                     /codon_start=1
                     /label=Cas6
                     /note="type I-F CRISPR-associated endoribonuclease 
                     Cas6/Csy4 [Vibrio cholerae]"
                     /translation="MKWYYKTITFLPELCNNESLAAKCLRVLHGFNYQYETRNIGVSFP
                     LWCDATVGKKISFVSKNKIELDLLLKQHYFVQMEQLQYFHISNTVLVPEDCTYVSFRRC
                     QSIDKLTAAGLARKIRRLEKRALSRGEQFDPSSFAQKEHTAIAHYHSLGESSKQTNRNF
                     RLNIRMLSEQPREGNSIFSSYGLSNSENSFQPVPLI"
     CDS             8577..9269
                     /codon_start=1
                     /label=TnsA
                     /note="TnsA endonuclease N-terminal domain-containing 
                     protein [Vibrio]"
                     /translation="MATSLPTPSAITTSALEYAFHTPARNLTKSRGKNIHRYVSVKMSK
                     RITVESTLECDACYHFDFEPSIVRFCAQPIRFLYYLNGQSHSYVPDFLVQFDTNEFVLY
                     EVKSAYAKNKPDFDVEWEAKVKAATELGLELELVEESDIRDTVVLNNLKRMHRYASKDE
                     LNNVHNSLLKIIKYNGAQSARCLGEQLGLKGRTVLPILCDLLSRCLLDTRLDKPLSLES
                     RFELASYG"
     CDS             9262..11073
                     /codon_start=1
                     /label=TnsB
                     /translation="MAKKGFSSFHRKAVSSQDTLESIELVSSANCLESVTYQDISAFPE
                     TIAVEINFRLSILRFLARKCETIVAKSIEPHRVELQQNYSRKIPSAITIYRWWLAFRKS
                     DYNPISLAPNIKDRGNRETKVSTVVDSIMEQAVERVISGRKVNVSSAYKRVRRKVRQYN
                     LTHGTKYTYPKYESVRKRVKKKTPFELLAAGKGERVAKREFRRMGKKILTSSVLERVEI
                     DHTVVDLFAVHEEYRIPLGRPWLTQLVDCYSKAVIGFYLGFEPPSYVSVSLALKNAIQR
                     KDDLISSYESIENEWLCYGIPDLLVTDNGKEFLSKAFDQACESLLINVHQNKVETPDNK
                     PHVERNYGTINTSLLDDLPGKSFSQYLQREGYDSVGEATLTLNEIREIYLIWLVDIYHK
                     KPNQRGTNCPNVAWKKGCQEWEPEEFSGSKDELDFKFAIVDYKQLTKVGITVYKELSYS
                     NDRLAEYRGKKGNHKVQFKYNPECMAVIWVLDEDMNEYFTVNAIDYEYASRVSLWQHKY
                     NMKYQAELNSAEYDEDKEIDAEIKIEEIADRSIVKTNKIRARRRGARHQENSARAKSIS
                     NANPASIQKHEDEIVSADNDDWDIDYV"
     CDS             10655..10666
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     CDS             11085..12077
                     /codon_start=1
                     /label=TnsC
                     /note="Tn6677 Vibrio cholerae transposon TnsC (VchTnsC) 
                     [Vibrio cholerae]"
                     /translation="MSETREARISRAKRAFVSTPSVRKILSYMDRCRDLSDLESEPTCM
                     MVYGASGVGKTTVIKKYLNQNRRESEAGGDIIPVLHIELPDNAKPVDAARELLVEMGDP
                     LALYETDLARLTKRLTELIPAVGVKLIIIDEFQHLVEERSNRVLTQVGNWLKMILNKTK
                     CPIVIFGMPYSKVVLQANSQLHGRFSIQVELRPFSYQGGRGVFKTFLEYLDKALPFEKQ
                     AGLANESLQKKLYAFSQGNMRSLRNLIYQASIEAIDNQHETITEEDFVFASKLTSGDKP
                     NSWKNPFEEGVEVTEDMLRPPPKDIGWEDYLRHSTPRVSKPGRNKNFFE"
     terminator      12115..12162
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    12183..12307
                     /label=VchINT R-end
     CDS             12317..12973
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     misc_feature    complement(13013..13157)
                     /label=VchINT L-end
     CDS             complement(join(13493..14031,1..121))
                     /codon_start=1
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"