pCasCure-Rif vector (Cat. No.: V015527)

GenBank Map(.gb) Copy Sequence View Map

Basic Information

Note: pCasCure-Rif plasmid containing the Cas9 gene with an l-arabinose inducible promoter pBAD, the sgRNA with the synthetic J23119 promoter, and the sacB gene for plasmid self-curing. F and R denote the locations of primers used for the sgRNA construct.

Name:: pCasCure-Rif

Length:: 9764 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Chen L.

Promoter:: araBAD

References

Hao M, He Y, Zhang H, et al. CRISPR-Cas9-Mediated Carbapenemase Gene and Plasmid Curing in Carbapenem-Resistant Enterobacteriaceae. Antimicrob Agents Chemother. 2020;64(9):e00843-20. Published 2020 Aug 20. doi:10.1128/AAC.00843-20

pCasCure-Rif vector (Cat. No.: V015527) Sequence

LOCUS       62056_4905        9764 bp DNA     circular SYN 26-MAY-2020
DEFINITION  Cloning vector pCasCure-Rif, complete sequence.
ACCESSION   MT262893
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9764)
  AUTHORS   Chen L.
  TITLE     CRISPR/Cas9-mediated plasmid curation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9764)
  AUTHORS   Chen L.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-MAR-2020) Center for Discovery and Innovation, 
            Hackensack-Meridian Health, 340 Kingsland Street, Nutley, NJ 07110, 
            USA
REFERENCE   3  (bases 1 to 9764)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-MAR-2020) Center for Discovery and Innovation, 
            Hackensack-Meridian Health, 340 Kingsland Street, Nutley, NJ 07110, 
            USA"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /label=AmpR promoter
     primer_bind     579..595
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(714..2132)
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        2518..2552
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        2570..2645
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2678..2694)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2707..6810)
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     promoter        complement(6838..7122)
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     CDS             7149..8024
                     /label=araC
                     /note="L-arabinose regulatory protein"
     primer_bind     complement(8151..8167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8175..8191)
                     /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)."
     promoter        complement(8199..8229)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8244..8265)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(8553..9141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9312..9764)
                     /codon_start=1
                     /transl_table=11
                     /product="Arr"
                     /label=Arr
                     /protein_id="QJX58432.1"
                     /translation="MVKDWIPISHDNYKQVQGPFYHGTKANLAIGDLLTTGFISHFEDG
                     RILKHIYFSALMEPAVWGAELAMSLSGLEGRGYIYIVEPTGPFEDDPNLTNKRFPGNPT
                     QSYRTCEPLRIVGVVEDWEGHPVELIRGMLDSLEDLKRRGLHVIED"

This page is informational only.