LOCUS       Exported               14164 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Targeting vector pTgKCNQ2 DNA, complete sequence.
ACCESSION   AB535096
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14164)
  AUTHORS   Tomonoh Y, Deshimaru M, Araki K, Miyazaki Y, Arasaki T, Tanaka Y, 
            Kitamura H, Mori F, Wakabayashi K, Yamashita S, Saito R, Itoh M, 
            Uchida T, Yamada J, Migita K, Ueno S, Kitaura H, Kakita A, Lossin C,
            Takano Y, Hirose S.
  TITLE     The kick-in system: a novel rapid knock-in strategy
  JOURNAL   PLoS ONE 9 (2), E88549 (2014)
  PUBMED    24586341
REFERENCE   2  (bases 1 to 14164)
  AUTHORS   Deshimaru M, Miyazaki Y, Arasaki T, Araki K, Hirose S.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-NOV-2009) Contact:Masanobu Deshimaru Fukuoka 
            University, Department of Chemistry, Faculty of Science; Nanakuma 
            8-19-1, Jonan-ku, Fukuoka, Fukuoka 814-0180, Japan
REFERENCE   3  (bases 1 to 14164)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..14164
                     /organism="Targeting vector pTgKCNQ2"
                     /mol_type="other DNA"
                     /note="Plasmid vector used for tergeted integration of 
                     sequence elements, including lox71, FRT, and lox2272 and 
                     PGK-Neo gene, into voltage-dependent potassium channel 
                     gene, Kcnq2, in mouse ES cell for following introduction of
                     a mutation by means of Cre recombinase-mediated 
                     lox-specific recombination."
                     /db_xref="taxon:696371"
     misc_feature    1..673
                     /note="sequence derived from backbone plasmid, pBluescript 
                     II SK+"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    674..2301
                     /standard_name="DT-A cassette"
                     /product="diphtheria toxin A"
                     /function="negative selection marker cassette"
                     /label=DT-A cassette
                     /note="DT-A cassette as a negative selection marker, which 
                     is removed through homologous recombination and functions 
                     for removal of cells with non-homologous recombination; 
                     This cassette consists of tk gene MC1 promoter (674-944), 
                     diphtheria toxin coding region (952-1608), intronic 
                     regulatory seqence from SV40 T antigen gene (1619-1709), 
                     and polyadenylation signal sequence from rabbit beta globin
                     gene."
     CDS             952..1608
                     /codon_start=1
                     /gene="DT-A"
                     /product="diphtheria toxin A"
                     /label=DT-A
                     /protein_id="BAI50644.1"
                     /translation="MDPDDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQG
                     NYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIK
                     KELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVE
                     LEINFETRGKRGQDAMYEYMAQACAGNRVRRSLCEGTLLLWCDIIGQTTYRDLKL"
     gene            952..1608
                     /gene="DT-A"
                     /label=DT-A
     CDS             961..1533
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          1610..1675
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     polyA_site      1888..1943
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     gene            2316..8090
                     /gene="Kcnq2"
                     /label=Kcnq2
     misc_feature    2316..7483
                     /gene="Kcnq2"
                     /standard_name="long arm"
                     /function="long arm for homologous recombination"
                     /label=long arm
                     /note="Kcnq2 sequence stretching from intron 1 to intron 5,
                     which induce recombination at the homologous genomic region
                     in mouse ES cell"
     misc_feature    7484..7517
                     /gene="Kcnq2"
                     /standard_name="lox71 element"
                     /function="left end-modified lox sequence"
                     /label=lox71 element
                     /note="modified lox sequence which can be recombined with 
                     loxKR3 element by Cre-mediated specific recombination."
     protein_bind    7484..7517
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    7518..8090
                     /gene="Kcnq2"
                     /note="Kcnq2 genomic segment consisting of exon 6 and 
                     flanking intronic sequence, which can be exchanged with the
                     mutated segment via Cre-lox mediated specific 
                     recombination."
     misc_feature    8091..10138
                     /standard_name="PGK-Neo cassette"
                     /product="neomycin phosphotransferase"
                     /function="positive selection marker cassette"
                     /label=PGK-Neo cassette
                     /note="This cassette consists of PGK promoter-driven 
                     neomycin resistant gene (8167-8713) and transcription 
                     termination/polyadenylation signal sequence from PGK gene 
                     (9745-10009), as well as a lox2272 element (9669-9702) for 
                     Cre-mediated recombination and two FRT elements (8167-8266,
                     10025-10124) for Flp-mediated removal of the marker gene."
     protein_bind    8093..8126
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(8183..8230)
                     /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)."
     promoter        8301..8801
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             8815..9618
                     /codon_start=1
                     /gene="Neo"
                     /product="neomycin phosphotransferase"
                     /label=Neo
                     /protein_id="BAI50645.1"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            8815..9618
                     /gene="Neo"
                     /label=Neo
     protein_bind    9669..9702
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     protein_bind    complement(10041..10088)
                     /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)."
     misc_feature    10145..11939
                     /gene="Kcnq2"
                     /standard_name="short arm"
                     /function="short arm for homologous recombination"
                     /label=short arm
                     /note="Kcnq2 sequence stretching from intron 6 to intron 8,
                     which induces recombination at the homologous genomic 
                     region in mouse ES cell"
     gene            10145..11939
                     /gene="Kcnq2"
                     /label=Kcnq2
     misc_feature    11940..14164
                     /note="sequence derived from backbone plasmid, pBluescript 
                     II SK+"
     promoter        complement(11976..11994)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12015..12031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    12039..12055
                     /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)."
     promoter        complement(12063..12093)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12108..12129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(12417..13005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(13176..14036)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(14037..14141)
                     /gene="bla"
                     /label=AmpR promoter