pUC18-mini-Tn7T-Plac-dCas9 (Plac) vector (V000100)

MSDS

Basic Vector Information

Vector Name:: pUC18-mini-Tn7T-Plac-dCas9 (Plac)

Antibiotic Resistance:: Ampicillin

Length:: 9404 bp

Type:: Bacterial Expression, CRISPR, Synthetic Biology

Replication origin:: ori

Selection Marker:: Gentamicin

Copy Number:: High Copy

pUC18-mini-Tn7T-Plac-dCas9 (Plac) 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 ℃.

pUC18-mini-Tn7T-Plac-dCas9 (Plac) vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       40924_44998        9404 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Tn7 integrating plasmid with S. pasteurianus dCas9 expressed from 
            the Plac promoter for expression in P. aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9404)
  AUTHORS   Tan SZ, Reisch CR, Prather KLJ
  TITLE     A Robust CRISPR Interference Gene Repression System in Pseudomonas.
  JOURNAL   J Bacteriol. 2018 Mar 12;200(7). pii: JB.00575-17. doi: 
            10.1128/JB.00575-17. Print 2018 Apr 1.
  PUBMED    29311279
REFERENCE   2  (bases 1 to 9404)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9404)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J
            Bacteriol."; date: "2018-03-12"; pages: " 10.1128/JB.00575-17. Print
            2018 Apr 1"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     534..550
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      578..672
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      775..861
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    907..954
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(1087..1617)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(1806..1834)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    1875..1922
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(1968..3047)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3048..3125)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        3355..3385
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3393..3409
                     /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)."
     CDS             3461..3487
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     terminator      6981..7007
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     mobile_element  complement(7099..7264)
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      complement(7534..8122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8296..9153)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(9154..9258)
                     /label=AmpR promoter
     primer_bind     9326..9344
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(9382..9404)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"

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