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ID   PHP45OMKM  preliminary; circular DNA; SYN; 4500 BP.
AC   K02163; ATCC37653;
DT   01-JUL-1993 (Rel. 7, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
DE   Broad host range/E.coli plasmid vector pHP45omega-Km - incomplete, m.
KW   cloning vector.
OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
RN   [1]
RC   pHP45omega-Km from pHP45omega & Tn5
RC   pHP45omega-Tc from pHP45omega & pBR322
RC   pHP45omega-Hg from pHP45omega & pME495
RC   pHP45omega-Cm from pHP45omega & pKT210
RC   pKT254omega from pKT254 & pJF70
RC   pKT254omega-Ap from pKT254omega & pKT235
RA   Fellay R., Frey J., Krisch H.;
RT   "Interposon mutagenesis of soil and water bacteria: a family of
RT   DNA fragments designed for in vitro insertional mutagenesis of
RT   gram-negative bacteria";
RL   Gene 52:147-154(1987).
RN   [2]
RP   1-155
RC   pHP45 from pBR322 & linker
RC   pHP45-omega from pHP45 & pKTH604 & R100.1, omega fragment
RC   [pCK1, pCK2 from T4 gene 32 & R100.1, omega/aadA gene]
RC   pRU876 from spec gene & terminators
RA   Prentki P., Krisch H.M.;
RT   "In vitro insertional mutagenesis with a selectable DNA fragment";
RL   Gene 29:303-313(1984).
CC   [2] describes a procedure combining the advantages of in vitro DNA
CC   linker mutagenesis with those of in vivo transposition mutagenesis.
CC   Insert the symmetrical 2.0-kb omega fragment, with an
CC   antibiotic resistance gene flanked by short inverted repeats
CC   carrying translation termination signals and synthetic polylinkers,
CC   into a linearized plasmid.  The omega fragment
CC   terminates RNA and protein synthesis prematurely, allowing the
CC   definition and mapping of both transcription and translation units.
CC   Because of its symmetry, it can be inserted in either direction.
CC   Cut pHP45omega with BssHII + SphI, removing the spcR/strR gene,
CC   and make the ends blunt. Ligate this to a blunt-end Tn5 1.5-kb
CC   HindIII-SalI fragment, containing the kanamycin resistance gene. [1]
CC   Because resistance levels may vary, it is important to determine the
CC   proper antibiotic concentration for each strain or isolate. [1]
CC   This is one of a series (ATCC37653-ATCC37657) of vectors for in vitro
CC   mutagenesis (via the omega interposon) of gram-negative bacteria. [1]
CC   Restriction digests of the clone give the following sizes (kb):
CC   EcoRI--2.35, 2.35; HindIII--2.4, 2.3; SalI--4.7. (ATCC staff)
CC   Medium is 1227 LB plus ampicillin.
CC   NCBI gi: 209010
CC   NM (pHP45omega-Km)
CC   CM (no)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   HO (E.coli C600)(broad host range)(E.coli)
CC   CP ()
CC   FN (cloning)
CC   SE ()
CC   PA (pBR322)(pKTH604)(R100.1 omega element)
CC   BR (pHP45omega-Cm)(pHP45omega-Hg)(pHP45omega-Tc)
CC   OF ()
CC   OR ()
FH   Key             Location/Qualifiers
FT   misc_feature    0..0
FT                   /note="1. pBR322 HindIII 4361bp 30..30
FT                   2. phage T4 HindIII-HindIII 2600bp, gene 32/59/33
FT                   -> pKSK12A 7000bp
FT                   1. pKSK12A remove small EcoRI-HindIII, 5' gene 32
FT                   -> pMJK4-18
FT                   1. pBR322 EcoRI 4361bp 4360..4360
FT                   2. linker SmaI/XmaI-BamHI-EcoRI 20bp
FT                   -> plasmid 4380bp
FT                   1. plasmid remove PvuII-HindIII 2039bp 30..2069,
FT                   \ tet gene/2341bp
FT                   :fill in [1 bp deleted]
FT                   -> pHP45 2340bp [unique HindIII, unique BamHI]
FT                   1. pHP45 2340bp
FT                   2. pKTH604 BamHI-HindIII 21bp, synthetic terminator
FT                   3. pMJK4-18 BstNI-HindIII 118bp, phage T4 gene 32 term
FT                   fill in:
FT                   BamHI linker 6bp ggatcc:
FT                   fill in:
FT                   4. R100.1 PvuII-HindIII 1700bp, omega spec/strep genes
FT                   \ #M60473
FT                   -> pHP45omega 4800bp
FT                   1. pHP45omega remove BssHII-SphI 1700bp,
FT                   \ spec/strep/3100bp
FT                   blunt end:blunt end
FT                   2. Tn5 HindIII-SalI 1489bp 1196..2685, kan gene
FT                   blunt end:blunt end
FT                   -> pHP45omega-Km 4500bp"
FT   misc_recomb     7..8
FT                   /note="omega inverted repeat end/T4 gene 32 start"
FT   misc_recomb     124..125
FT                   /note="T4 gene 32 end/pKTH604 fragment start"
FT   misc_recomb     139..140
FT                   /note="pKTH604 fragment end/polylinker start"
FT   misc_binding    0..0
FT                   /note="MCS unique EcoRI-SmaI-BamHI-HindIII or
FT                   HindIII-BamHI-SmaI-EcoRI"
FT   rep_origin      0..0
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322)"
FT   CDS             0..0
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp)"
FT   CDS             0..0
FT                   /note="ANT E. coli kanamycin resistance gene (kan)"
SQ   Sequence 155 BP; 46 A; 31 C; 26 G; 52 T; 0 other;
     gatccggtgg atgacctttt gaatgacctt taatagatta tattactaat taattgggga
     ccctagaggt cccctttttt attttaaaaa ttttttcaca aaacggttta caagcataaa
     gcttgctcaa tcaatcaccg gatccccggg aattc