gRNA_Cloning Vector vector (V007529)

Basic Vector Information

      • Vector Name:
      • gRNA_Cloning Vector
      • Antibiotic Resistance:
      • Kanamycin
      • Length:
      • 3914 bp
      • Type:
      • Mammalian Expression, CRISPR
      • Cloning Method:
      • TOPO Cloning
      • 5' Primer:
      • T7

gRNA_Cloning Vector vector Vector Map

gRNA_Cloning Vector3914 bp60012001800240030003600pBR322ori-FL4440CAP binding sitelac promoterlac operatorM13 revSP6 promoterattB2LKO.1 5'hU6-FT7M13 ForwardccdBNeoR/KanRKozak sequenceori

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 ℃.

gRNA_Cloning Vector vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                3914 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  An empty gRNA expression vector, used to create a gRNA to a specific
            sequence..
ACCESSION   .
VERSION     .
KEYWORDS    gRNA_Cloning Vector
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3914)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE,
            Church GM
  TITLE     RNA-Guided Human Genome Engineering via Cas9.
  JOURNAL   Science. 2013 Jan 3.
  PUBMED    23287722
REFERENCE   2  (bases 1 to 3914)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2013 Jan 3."
FEATURES             Location/Qualifiers
     source          1..3914
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     380..399
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     633..650
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    767..788
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        803..833
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    841..857
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     primer_bind     846..868
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     865..881
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     865..881
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        899..917
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     899..916
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1002..1026
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1133..1152)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1303..1323)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1462..1481)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1463..1481)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1488..1505)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1488..1504)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1497..1519)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1642..1944
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1823..1842
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2293..3087
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2347..2366)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2957..2976
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3287..3296
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3293..3667
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      join(3805..3914,1..479)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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