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ID   PAL4000    preliminary; circular DNA; SYN; 13000 BP.
AC   ATCC77192;
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 pAL4000 - incomplete.
KW   cloning vector.
OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
RN   [1]
RC   pAL37 from RK2
RC   pAL13 from pBR322 & linker
RC   pAL14 from pAL13 & pKW102, luc gene
RC   [pAD9 from terminator]
RC   pAL1 from pUC19 & pAD9, terminator
RC   pAL6 from pAL1 & pTJS37, tet gene
RC   pAL23 from pAL14 & pAL6
RC   pAL27 from pAL23 & linker
RC   pAL4000 from pAL27 & RSF1010
RC   pAL20 from pAL13 & pUC19 & Tn9 cat gene
RC   pUCD2B from pUC7 & RK2, ori
RC   pAL20RK2p from pAL20 & pUCD2B & pAL6 & linker
RC   pAL4000RK2p from pAL4000 & pAL20RK2p
RA   Greener A., Lehman S.M., Helinski D.R.;
RT   "Promoters of the broad host range plasmid RK2: analysis of
RT   transcription (initiation) in five species of gram-negative
RT   bacteria";
RL   Genetics 130:27-36(1992).
RN   [2]
RC   pKW102 from luc gene
RA   Woods K.;
RT   ;
RL   Unpublished (1992).
RN   [3]
RC   pTJS53 from pUC8 & RK2
RC   pTJS65 from pUC8 & RK2
RC   pTJS66 from pTJS53 & RK2
RC   pTJS67 from RK2
RC   pTJS75 from RK2
RC   pTJS81 from pUC7 & RK2
RC   pTJS87 from RK2
RC   pTJS94 from pCT88 & pTJS65
RC   pTJS100 from RK2
RC   pUC9CM from pUC9 & cat gene
RC   pTJS108 from pUC9CM & pBR322
RC   pTJS111 from RK2
RC   pTJS112 from RK2
RC   pTJS117 from RK2
RC   pTJS124 from RK2
RC   pRK2178 from RK2
RC   pTJS129 from pRK2178 & pUC9CM
RC   pTJS130 from RK2
RC   pTJS133 from RK2
RC   pTJS175 from RK2
RC   pTJS194 from pTJS108 & pTJS129
RC   pTJS210 from RK2
RA   Schmidhauser T.J., Helinski D.R.;
RT   "Regions of broad host range plasmid RK2 involved in replication
RT   and stable maintenance in nine species of Gram-negative bacteria";
RL   J. Bacteriol. 164:446-455(1985).
RN   [4]
RC   pHS40 from RP4
RA   Schwab H., Saurugger P.N., Lafferty R.M.;
RT   "Occurrence of deletion plasmids at high rates after conjugative
RT   transfer of the plasmids RP4 and RK2 from Escherichia coli to
RT   Alcaligenes eutrophus H16";
RL   Arch. Microbiol. 136:140-146(1983).
RN   [5]
RC   pCT88 from p15A
RA   Thomas C.M.;
RT   "Complementation analysis of replication and maintenance functions
RT   of braod host range plasmids RK2 and RP1";
RL   Plasmid 5:277-291(1981).
RN   [6]
RC   pDG4 from RK2 & ColE1
RC   pDG5 from RK2 & ColE1
RA   Guiney D.G., Yakobson E.;
RT   "Location and nucleotide sequence of the transfer origin of the
RT   broad host range plasmid RK2";
RL   Proc. Natl. Acad. Sci. U.S.A. 80:3595-3598(1983).
RN   [7]
RC   pTJS26 from RK2, tet gene
RC   pTJS29A from ColE1, tet gene
RC   pTJS37 from RK2, tet gene
RC   pTJS50 from RK2, tet gene
RA   Schmidhauser T.J., Filutowicz M., Helinski D.R.;
RT   "Replication of derivatives of the braod host range plasmid RK2 in
RT   two distantly related bacteria";
RL   Plasmid 9:325-330(1983).
CC   Promoter-cloning vector permitting visual detection of activity using
CC   the promoterless luciferase gene as the reporter. [1]
CC   The order of the major features in this plasmid is: rpoC
CC   rho-independent transcription terminator - EcoRI/MCS/PstI -
CC   translation terminator - luc - BglII - RSF1010 - tetR.
CC   Not self transmissible, but mobilizable by the transfer functions of
CC   RK2.
CC   Was tested in Pseudomonas aeruginosa PAO1161, Acinetobacter
CC   calcoaceticus BD413, Rhizobium meliloti 102F34nal, and Agrobacterium
CC   tumefaciens A18.
CC   Restriction digests of the clone give the following sizes (kb):
CC   BamHI--12.5; EcoRI--9.0, 2.3, 1.2, 0.6, 0.25; PstI/BglII--10.0, 2.4.
CC   (ATCC staff)
CC   Medium is 1273 LB plus tetracycline.
CC   NM (pAL4000)
CC   CM (no)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   HO (E.coli)(broad host range)(Gram-negative bacteria)
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. pBR322 BamHI 4361bp 376..376
FT                   fill in
FT                   SalI 4361bp 652..652
FT                   fill in
FT                   BglII linker 8bp cagatctg
FT                   -> pAL13 4369bp
FT                   1. firefly, luc gene
FT                   -> pKW102
FT                   1. pAL13 remove HindIII-EcoRV 158bp 30..188, 4211bp
FT                   2. pKW102 HindIII-SmaI, firefly luc gene
FT                   -> pAL14
FT                   1. terminator
FT                   -> pAD9
FT                   1. pUC19 EcoRI 2686bp 397..397, MCS
FT                   2. pAD9 EcoRI-EcoRI, terminator
FT                   -> pAL1
FT                   1. RK2, tet gene
FT                   -> pTJS37
FT                   1. pAL1 EcoRI
FT                   2. pTJS37 XhoII-StuI, tet gene
FT                   EcoRI linker 6bp gaattc:EcoRI linker 6bp gaattc
FT                   -> pAL6
FT                   1. pAL14 remove small HindIII-EcoRI
FT                   2. pAL6 HindIII-EcoRI, tet/term/MCS
FT                   -> pAL23 5100bp
FT                   1. pAL23 HindIII 5100bp
FT                   fill in
FT                   2. linker 12bp ctagctagctag, terminator
FT                   -> pAL27 5100bp
FT                   1. pAL27 BglII-EcoRI 5100bp,
FT                   \ tet/term/MCS/term/luc/pBR322
FT                   2. RSF1010 remove PstI-PstI 800bp 7773..8573, 7884bp
FT                   EcoRI linker 6bp gaattc:BglII linker 6bp agatct
FT                   -> pAL4000 13000bp"
FT   rep_origin      0..0
FT                   /note="ORI E. coli IncQ plasmid RSF1010 oriV and oriT"
FT   CDS             0..0
FT                   /note="ANT E. coli tetracycline resistance gene (tet)"
FT   terminator      0..0
FT                   /note="TER"
FT   misc_binding    0..0
FT                   /note="MCS EcoRI-SacI-KpnI-BamHI-XbaI-PstI"
FT   misc_binding    0..0
FT                   /note="SIT unique SacI-KpnI-BamHI-XbaI-PstI"
FT   terminator      0..0
FT                   /note="TER"
FT   CDS             0..0
FT                   /note="GEN firefly luciferase gene (luxA);
FT                   reporter gene"
FT   misc_feature    0..0
FT                   /note="from pBR322 185..651"
SQ   Sequence 1 BP; 0 A; 0 C; 0 G; 0 T; 1 other;