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ID   PGT45A     preliminary; circular DNA; SYN; 9500 BP.
AC   X66900; X66902; S37047; X66901; S37046; X66903; S37048; ATCC77329;
DT   03-FEB-1994 (Rel. 8, Created)
DT   01-APR-1995 (Rel. 11, Last updated, Version 1)
DE   Vertebrate/E.coli plasmid vector pGT4.5A - incomplete, En-2/lacZ.
KW   cloning vector.
OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
RN   [1]
RC   from pGT4.5
RA   Skarnes W.C., Auerbach B.A., Joyner A.L.;
RT   "A gene trap approach in mouse embryonic stem cells: the lacZ
RT   reporter is activated by splicing, reflects endogenous gene
RT   expression, and is mutagenic in mice";
RL   Genes Dev. 6:903-918(1992).
RN   [2]
RC   p3LSN from pMC1871 & piVX & pMoTN & mouse hsp68 gene promoter
RC   p6LSN from pMC1871 & piVX & pMoTN
RC   fragment from human beta-actin promoter & neo gene
RC   pGT4.5 from pMC1871 & mouse En-2 homeobox exon & fragment
RA   Gossler A., Joyner A.L., Rossant J., Skarnes W.C.;
RT   "Mouse embryonic stem cells and reporter constructs to detect
RT   developmentally regulated genes";
RL   Science 244:463-465(1989).
RN   [3]
RC   pUC8::HSV-TK from pUC8 & HSV tk gene promoter
RC   pMoTN from pUC8::HSV-TK & pBR322 & Tn5, neo gene
RC   NeoF101 from polyoma virus mutant & SV(X)
RA   Magli M.C., Dick J.E., Huszar D., Bernstein A., Phillips R.A.;
RT   "Modulation of gene expression in multiple hematopoietic cell
RT   lineages following retroviral vector gene transfer";
RL   Proc. Natl. Acad. Sci. U.S.A. 84:789-793(1987).
RN   [4]
RC   SV(X) from SV40
RC   NEOmu from SV(X) & Ig heavy chain enhancer
RA   Dick J.E., Magli M.C., Huszar D., Phillips R.A., Bernstein A.;
RT   "Introduction of a selectable gene into primitive stem cells
RT   capable of long-term reconstitution of the hemopoietic system of
RT   W/Wv mice";
RL   Cell 42:71-79(1985).
RN   [5]
RC   MLV.NEO.1 from MLV & neo gene
RA   Joyner A.L., Bernstein A.;
RT   "Retrovirus transduction: generation of infectious retroviruses
RT   expressing dominant and selectable genes is associated with in
RT   vivo recombination and deletion events";
RL   Mol. Cell. Biol. 3:2180-2190(1983).
CC   Deposited by: Alexandra L. Joyner
CC   Gene trap vector designed to identify and mutate endogenous mammalian
CC   genes by integration and production of a fusion protein with the lacZ
CC   reporter gene. Can also be used to monitor endogenous gene expression.
CC   [1]
CC   Identified cDNA sequences spanning the lacZ splice junction can be
CC   cloned using the published sequence of the En-2 splice acceptor and
CC   the rapid amplification of cDNA ends (RACE) protocol. [1]
CC   Derived from pGT4.5 by replacing the En-2 polyadenylation signal with
CC   an SV40 polyadenylation signal. (personal communication)
CC   The order of the major features in this plasmid is:  pUC18 - HindIII -
CC   En-2 intron - splice acceptor - En-2 homeobox exon - lacZ - SV40
CC   polyadenylation - human beta-actin promoter - neoR - SV40
CC   polyadenylation. [2]
CC   Restriction digests of the clone give the following sizes (kb):
CC   BamHI--3.8, 3.3, 1.8, 0.45, 0.2; HindIII--9.5; XbaI--4.6, 3.1, 1.0,
CC   0.8. (ATCC staff)
CC   Medium is 1227 LB plus ampicillin.
CC   NCBI gi: 403021
CC   NM (pGT4.5A)
CC   CM (no)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   HO (E.coli)(E.coli HB101)(vertebrate cells)
CC   CP ()
CC   FN (cloning)
CC   SE ()
CC   PA ()
CC   BR ()
CC   OF ()
CC   OR ()
FH   Key             Location/Qualifiers
FT   misc_feature    0..0
FT                   /note="1. pMC1871 remove EcoRI-SstI, MCS/lacZ gene
FT                   2. mouse HindIII, 5' En-2 gene
FT                   blunt end
FT                   EcoRI-SstI 1800bp, 5' En-2 gene
FT                   -> plasmid
FT                   1. plasmid
FT                   2. human beta-actin promoter
FT                   3. neo gene
FT                   -> plasmid2
FT                   1. plasmid2 remove small BglII-SstI, neo gene
FT                   2. mouse BglII-SstI 2700bp, 3' En-2 gene
FT                   -> pGT4.5 [HindIII at both ends of reporter gene]"
FT   rep_origin      0..0
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322)"
FT   CDS             0..0
FT                   /note="GEN E. coli beta-galactosidase gene (lacZ');
FT                   reporter gene"
FT   CDS             0..0
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp)"
FT   CDS             47..>271
FT                   /note="GEN E. coli En-2/lacZ junction; 2 stops
FT                   when translation attempted, frame 2, code 0"
SQ   Sequence 271 BP; 75 A; 85 C; 68 G; 43 T; 0 other;
     ccagcaacca gtaacctctg ccctttctcc tccatgacaa ccaggtccca ggtcccgaaa
     accaaagaag aagaacccta acaaagagga caagcggcct cgcacagcct tcactgctga
     gcagctccag aggctcaagg ctgagtttca gaccaacagg tacctgacag agcagcggcg
     ccagagtctg gcacaggagc tcggtacccg gaagatctgg actctagagg atcccgtcgt
     tttacaacgt cgtgactggg aaaaccctgg c