pJMP1 vector (V006809) Gene synthesis in pJMP1 backbone

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Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V006809	pJMP1	In stock, instant shipping

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

Basic Vector Information

pJMP1 is a core plasmid designed for CRISPR interference (CRISPRi) in Bacillus subtilis, engineered to express the nuclease-deactivated Cas9 (dCas9) under the control of a xylose-inducible promoter (Pxyl) and carry an erythromycin resistance gene (Ermᵣ) for selection. pJMP1 enables dose-dependent and specific transcriptional repression of target genes when paired with sgRNA-expressing plasmids like pJMP2 or pJMP3. pJMP1 integrates into lacA locus.

Vector Name:: pJMP1

Antibiotic Resistance:: Ampicillin

Length:: 12078 bp

Type:: Bacterial Expression, CRISPR

Replication origin:: ori

Selection Marker:: Bacillus subtilis erythromycin/lincomycin marker

Copy Number:: High Copy

Promoter:: xylA

Cloning Method:: Restriction Enzyme

5' Primer:: TCCTTTGTTTATCCACCGAAC

3' Primer:: GCCTCGTGATACGCCTATTT

Growth Strain(s):: DH10B

pJMP1 vector Map

Copy Sequence View Map

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

Peters JM, Colavin A, Shi H, Czarny TL, Larson MH, Wong S, Hawkins JS, Lu CHS, Koo BM, Marta E, Shiver AL, Whitehead EH, Weissman JS, Brown ED, Qi LS, Huang KC, Gross CA. A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria. Cell. 2016 Jun 2;165(6):1493-1506. doi: 10.1016/j.cell.2016.05.003. Epub 2016 May 26. PMID: 27238023; PMCID: PMC4894308.

pJMP1 vector Sequence

LOCUS       Exported               12078 bp DNA     circular SYN 09-DEC-2025
DEFINITION  Bacillus subtilis dCas9 expression vector; integrates into 
            lacA/ganA.
ACCESSION   .
VERSION     .
KEYWORDS    pJMP1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12078)
  AUTHORS   Peters JM, Colavin A, Shi H, Czarny TL, Larson MH, Wong S, Hawkins 
            JS, Lu CH, Koo BM, Marta E, Shiver AL, Whitehead EH, Weissman JS, 
            Brown ED, Qi LS, Huang KC, Gross CA
  TITLE     A Comprehensive, CRISPR-based Functional Analysis of Essential Genes
            in Bacteria.
  JOURNAL   Cell. 2016 Jun 2;165(6):1493-506. doi: 10.1016/j.cell.2016.05.003. 
            Epub 2016 May 26.
  PUBMED    27238023
REFERENCE   2  (bases 1 to 12078)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 12078)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2016.05.003"; journalName: "Cell"; date: "2016-06-2-
            2"; volume: "165"; issue: "6"; pages: "1493-506"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..12078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1085..1276
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    1378..1518
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1533..1550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      1681..2282
                     /label=ColE1 replication origin
                     /note="High-copy number plasmid replication origin derived
                     from ColE1, commonly used in molecular cloning vectors for 
                     efficient DNA replication in bacterial hosts; id: 4341693"
     CDS             complement(2463..3323)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        3324..3454
                     /label=AmpR promoter
                     /note="Regulatory region that initiates transcription of
                     the ampicillin resistance gene in bacterial plasmids; id: 
                     3672594"
     gene            complement(3539..4038)
                     /label='lacA
                     /note="'lacA"
     regulatory      4045..4126
                     /label=Lambda phage transcription terminator
                     /note="Transcription termination site from bacteriophage
                     lambda that signals RNA polymerase to stop transcription 
                     and release the RNA transcript.; id: 3977482"
     CDS             complement(4138..5262)
                     /codon_start=1
                     /product="XylR is a repressor of xylose operon regulating 
                     promoter Pxyl."
                     /label=Bacillus megaterium xylR
                     /translation="MNQKLILDEILKNSPVSRATLSEITGLNKSTVSSQVNTLLEKDFI
                     FEIGAGQSRGGRRPVMLVFNKNAGYSIGIDIGVDYLNGILTDLEGNIILEKTSDLSSSS
                     ASEVKEILFALIHGFVTHMPESPYGLVGIGICVPGLVDRHQQIIFMPNLNWNIKDLQFL
                     IESEFNVPVFVENEANAGAYGEKVFGMTKNYENIVYISINIGIGTGLVINNELYKGVQG
                     FSGEMGHMTIDFNGPKCSCGNRGCWELYASEKALLASLSKEEKNISRKEIVERANKNDV
                     EMLNALQNFGFYIGIGLTNILNTFDIEAVILRNHIIESHPIVLNTIKNEVSSRVHSHLD
                     NKCELLPSSLGKNAPALGAVSIVIDSFLSVTPIS"
     regulatory      5305..5329
                     /label=xylR ribosome binding site cluster
                     /note="Multiple ribosome binding sites associated with the
                     xylR gene, facilitating translation initiation of xylose 
                     metabolic regulatory proteins; id: 3977299"
     promoter        5330..5440
                     /label=XylR promoter
                     /note="Regulatory region that controls transcription
                     initiation for genes responsive to the XylR transcriptional
                     regulator, commonly involved in xylose metabolism pathways;
                     id: 3977300"
     promoter        5376..5507
                     /label=Xylose-inducible promoter PxylA(+66)
                     /note="A strong, constitutive variant of the
                     xylose-inducible PxylA promoter from Bacillus subtilis, 
                     commonly used for controlled gene expression in 
                     Gram-positive bacteria.; id: 3977524"
     regulatory      5508..5529
                     /label=xylA ribosome binding site cluster
                     /note="A cluster of ribosome binding sites that facilitates
                     translation initiation for the xylA gene, enabling 
                     efficient production of xylose metabolic enzymes.; id: 
                     3977566"
     CDS             5578..9684
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     regulatory      9700..9779
                     /label=BBa_B0010 transcriptional terminator
                     /note="A transcriptional terminator sequence that signals
                     the end of transcription, preventing RNA polymerase from 
                     continuing downstream and ensuring proper gene expression 
                     boundaries.; id: 3687631"
     terminator      9788..9828
                     /label=Transcription Terminator Cluster
                     /note="A collection of transcription termination sequences 
                     including B0014, B0012, and T7t terminators that function 
                     to halt RNA polymerase and terminate transcription in 
                     genetic circuits.; id: 3687636"
     mobile_element  9897..11561
                     /label=pMutin Plasmid Insertion Element
                     /note="A pMutin plasmid-derived genetic element used for 
                     insertional mutagenesis, typically serving as a vector for 
                     gene disruption or modification in bacterial systems.; id: 
                     4011928"
     CDS             10380..11117
                     /codon_start=1
                     /product="23S rRNA (adenine(2058)-N(6))-methyltransferase"
                     /label=Erm
                     /translation="MNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTVYEIGTGKGHLTT
                     KLAKISKQVTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNKQRYKIVGSIPYH
                     LSTQIIKKVVFESHASDIYLIVEEGFYKRTLDIHRTLGLLLHTQVSIQQLLKLPAECFH
                     PKPKVNSVLIKLTRHTTDVPDKYWKLYTYFVSKWVNREYRQLFTKNQFHQAMKHAKVNN
                     LSTVTYEQVLSIFNSYLLFNGRK"
     regulatory      11281..11379
                     /label=rrnB T1 transcriptional terminator
                     /note="Rho-independent transcriptional terminator that
                     halts RNA polymerase transcription at the end of the 
                     Escherichia coli rrnB ribosomal RNA operon, forming a 
                     stable stem-loop structure in the RNA transcript.; id: 
                     4325218"
     terminator      11390..11420
                     /label=rrnB T2 transcription terminator
                     /note="Transcription termination signal from the E. coli 
                     ribosomal RNA operon rrnB that promotes RNA polymerase 
                     dissociation from DNA template; id: 4381029"
     terminator      11499..11533
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     gene            complement(11576..12075)
                     /label=lacA'
                     /note="lacA'"