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ID   PMMB66EH   preliminary; circular DNA; PRO; 8807 BP.
XX
AC   X15234; M28829; V00294; X15194; ATCC37620;
XX
DT   20-DEC-1989 (Rel. 4, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
XX
DE   Broad host range/E.coli plasmid vector pMMB66EH - complete.
XX
KW   cloning vector.
XX
OS   Pseudomonas
OC   Prokaryota; Bacteria; Gracilicutes; Scotobacteria;
OC   Facultatively anaerobic rods; Enterobacteriaceae.
XX
RN   [1]
RP   1-8807
RC   pMMB66EH
RA   Morales V.M.;
RT   ;
RL   Submitted (08-MAY-1989) on tape to the EMBL Data Library by:
RL   Morales V.M., Michigan Biotechnology Institute, 3900 Collins Road,
RL   Lansing MI 48909, USA.
XX
RN   [2]
RP   1-8807
RC   RP4, R751 from E. coli IncQ plasmids
RA   Morales V.M., Baeckman A., Bagdasarian M.;
RT   "Promiscuous plasmids of the IncQ group. Mode of replication and
RT   use for gene cloning in Gram-negative bacteria.";
RL   Pseudomonas. Biotransformations, Pathogenesis and ... 0:1-1(1990).
RL   Silver S., Chakrabarty A.M., Gunsalus R.P., Iglewski B.;
RL   (eds.), Academic Press, New York.
XX
RN   [3]
RC   pJF118EH, pJF118HE from pMMB22 & pKK223-3
RC   pMMB66EH from RSF1010 & pJF118EH
RC   pMMB66HE from pMMB66EH & pJF118HE
RC   pJF119EH from pJF118EH & M13mp18
RC   pJF119HE from pJF118HE & M13mp18
RC   pMMB67EH from pMMB66EH & M13mp18
RC   pMMB67HE from pMMB66HE & M13mp18
RC   pMS324a from R300B & RP4
RC   pFG101 series from pJF118EH & pMS324a
RC   pFG102 series from pJF118HE & pMS324a
RC   pWP101 series from pMMB66EH & pMS324a
RC   pWP102 series from pMMB66HE & pMS324a
RA   Furste J.P., Pansegrau W., Frank R., Blocker H., Scholz P.,
RA   Bagdasarian M., Lanka E.;
RT   "Molecular cloning of the plasmid RP4 primase
RT   region in a multi-host-range tacP expression vector";
RL   Gene 48:119-131(1986).
XX
RN   [4]
RC   pMMB66EH from RSF1010 & pJF118EH
RA   Morales V.M., Backman A., Bagdasarian M.;
RT   "Sequence of junctions of expression vector pMMB66EH";
RL   Nucleic Acids Res. 17:0-0(1989).
XX
CC   This sequence overlaps M28829 and V00294.
CC   Restriction digests of the clone give the following sizes (kb):
CC   AccI--3.9, 3.0, 1.9; BamHI--8.8; HindIII/PvuII--7.6, 1.3. (ATCC staff)
CC   Contains the following sites separated by (bp): EcoRI-764 -AhaIII-97
CC   -ScaI-112 -PvuI-2101 -AccI-1848 -ScaI-55 -AccI-2039 -BstEII-372
CC   -PvuII-93 -PvuII-94 -HpaI-320 -BstEII-182 -MluI-736 -EcoRI. [3]
CC   The sequence of RSF1010 is given in this reference. [4]
CC   A 1246 bp lacIQ HindIII fragment was inserted at XmaIII/BamHI of
CC   pKK223-3. The 2995 bp NruI/AhaIII fragment of the resulting plasmid
CC   was cloned between PstI (bp 7768) and PvuII (bp 1951) of RSF1010. [3]
CC   In the pMMB66 (ATCC 37620, 37621) and pMMB67 (ATCC 37622, 37623)
CC   vector series,the EH and HE in the name designate the orientation of
CC   the multiple cloning site to the tac promoter.
CC   (personal communication)
CC   An autoregulated high-level expression vector utilizing the tac
CC   promoter, rrnB terminators and lacIq for lac repression.
CC   Concentration of induced gene product may be regulated by varying the
CC   lac inducer concentration. [3]
CC   Can be mobilized by conjugative IncP helper plasmids [e.g. pRK2013
CC   (ATCC 37159), pUB307] into Klebsiella aerogenes, Proteus mirabilis,
CC   Pseudomonas aeruginosa, Serratia marcescens, and other gram-negative
CC   bacteria. [3]
CC   Medium is 1227 LB plus ampicillin.
CC   NM (pMMB66EH)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP (ATCC)
CC   HO (Gram-negative bacteria)(E.coli K-12)(E.coli HB101)(E.coli)
CC   HO (broad host range)
CC   CP (low)
CC   FN (cloning)
CC   SE (color blue/white)
CC   PA ()
CC   BR (pMMB190)(pMMB206)
CC   OF ()
CC   OR ()
XX
FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pMMB22 HindIII-HindIII 1247bp, lacIq gene
FT                   2. pKK223-3 remove BamHI-XmaIII 564bp 257..821
FT                   \ 4020bp [547bp]
FT                   -> pJF118EH 5267bp
FT                   1. RSF1010 remove PstI-PvuII 2754bp 7910..8684..1980,
FT                   \ 5930bp
FT                   2. pJF118EH NruI-AhaIII 2996bp, pKK223-3 NruI-? 856..?
FT                   -> pMMB66EH 8807bp"
FT   promoter        482..509
FT                   /note="PRO E. coli bla gene"
FT   CDS             552..1409
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp) (AA 1-286)"
FT   rep_origin      1850..2245
FT                   /note="ORI E. coli IncQ plasmid origin of vegetative
FT                   replication oriV"
FT   CDS             complement(2273..2554)
FT                   /note="GEN E. coli mobC gene
FT                   (conjugational DNA transfer) (AA 1-94)"
FT   promoter        2546..2576
FT                   /note="PRO E. coli promoter P1"
FT   promoter        2565..2595
FT                   /note="PRO E. coli promoter P3"
FT   rep_origin      2585..2672
FT                   /note="ORI E. coli IncQ plasmid origin of conjugative
FT                   DNA transfer oriT"
FT   promoter        complement(2587..2617)
FT                   /note="PRO E. coli promoter"
FT   CDS             2753..4879
FT                   /note="GEN E. coli repB/mobA gene (primase)
FT                   (AA 1-709)"
FT   CDS             3501..3911
FT                   /note="GEN E. coli mobB gene
FT                   (conjugational DNA transfer) (AA 1-137)"
FT   CDS             3911..4879
FT                   /note="GEN E. coli repB' gene (primase) (AA 1-323)"
FT   promoter        4884..4915
FT                   /note="PRO E. coli promoter P4"
FT   CDS             4943..5152
FT                   /note="GEN E. coli E gene (function unknown)
FT                   (AA 1-70)"
FT   CDS             5157..5360
FT                   /note="GEN E. coli cac gene
FT                   (regulates repA and repC gene expression) (AA 1-68)"
FT   CDS             5393..6229
FT                   /note="GEN E. coli repA gene (DNA helicase)
FT                   (AA 1-279)"
FT   CDS             6219..7067
FT                   /note="GEN E. coli repC gene (replication initiator
FT                   protein) (AA 1-283)"
FT   promoter        7231..7261
FT                   /note="PRO E. coli promoter P5"
FT   promoter        complement(8463..8492)
FT                   /note="PRO E. coli lacIq repressor gene"
FT   promoter        8736..8764
FT                   /note="PRO E. coli tac (trp and lac)"
FT   misc_binding    0..0
FT                   /note="MCS unique EcoRI-SmaI-BamHI-SalI-PstI-HindIII"
FT   rep_origin      0..0
FT                   /note="ORI E. coli RSF1010"
FT   CDS             0..0
FT                   /note="REP E. coli lacIq repressor gene"
XX
SQ   Sequence 8807 BP; 1904 A; 2577 C; 2631 G; 1695 T; 0 other;
     ttcccgggga tccgtcgacc tgcagccaag cttggctgtt ttggcggatg agagaagatt
     ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     tttagattga tttctgaaag cgaccaggtg ctcggcgtgg caagactcgc agcgaacccg
     tagaaagcca tgctccagcc gcccgcattg gagaaattct tcaaattccc gttgcacata
     gcccggcaat tcctttccct gctctgccat aagcgcagcg aatgccgggt aatactcgtc
     aacgatctga tagagaaggg tttgctcggg tcggtggctc tggtaacgac cagtatcccg
     atcccggctg gccgtcctgg ccgccacatg aggcatgttc cgcgtccttg caatactgtg
     tttacataca gtctatcgct tagcggaaag ttcttttacc ctcagccgaa atgcctgccg
     ttgctagaca ttgccagcca gtgcccgtca ctcccgtact aactgtcacg aacccctgca
     ataactgtca cgcccccctg caataactgt cacgaacccc tgcaataact gtcacgcccc
     caaacctgca aacccagcag gggcgggggc tggcggggtg ttggaaaaat ccatccatga
     ttatctaaga ataatccact aggcgcggtt atcagcgccc ttgtggggcg ctgctgccct
     tgcccaatat gcccggccag aggccggata gctggtctat tcgctgcgct aggctacaca
     ccgccccacc gctgcgcggc agggggaaag gcgggcaaag cccgctaaac cccacaccaa
     accccgcaga aatacgctgg agcgctttta gccgctttag cggcctttcc ccctacccga
     agggtggggg cgcgtgtgca gccccgcagg gcctgtctcg gtcgatcatt cagcccggct
     catccttctg gcgtggcggc agaccgaaca aggcgcggtc gtggtcgcgt tcaaggtacg
     catccattgc cgccatgagc cgatcctccg gccactcgct gctgttcacc ttggccaaaa
     tcatggcccc caccagcacc ttgcgccttg tttcgttctt gcgctcttgc tgctgttccc
     ttgcccgctc ccgctgaatt tcggcattga ttcgcgctcg ttgttcttcg agcttggcca
     gccgatccgc cgccttgttg ctccccttaa ccatcttgac accccattgt taatgtgctg
     tctcgtaggc tatcatggag gcacagcggc ggcaatcccg accctacttt gtaggggagg
     gcgcacttac cggtttctct tcgagaaact ggcctaacgg ccacccttcg ggcggtgcgc
     tctccgaggg ccattgcatg gagccgaaaa gcaaaagcaa cagcgaggca gcatggcgat
     ttatcacctt acggcgaaaa ccggcagcag gtcgggcggc caatcggcca gggccaaggc
     cgactacatc cagcgcgaag gcaagtatgc ccgcgacatg gatgaagtct tgcacgccga
     atccgggcac atgccggagt tcgtcgagcg gcccgccgac tactgggatg ctgccgacct
     gtatgaacgc gccaatgggc ggctgttcaa ggaggtcgaa tttgccctgc cggtcgagct
     gaccctcgac cagcagaagg cgctggcgtc cgagttcgcc cagcacctga ccggtgccga
     gcgcctgccg tatacgctgg ccatccatgc cggtggcggc gagaacccgc actgccacct
     gatgatctcc gagcggatca atgacggcat cgagcggccc gccgctcagt ggttcaagcg
     gtacaacggc aagaccccgg agaagggcgg ggcacagaag accgaagcgc tcaagcccaa
     ggcatggctt gagcagaccc gcgaggcatg ggccgaccat gccaaccggg cattagagcg
     ggctggccac gacgcccgca ttgaccacag aacacttgag gcgcagggca tcgagcgcct
     gcccggtgtt cacctggggc cgaacgtggt ggagatggaa ggccggggca tccgcaccga
     ccgggcagac gtggccctga acatcgacac cgccaacgcc cagatcatcg acttacagga
     ataccgggag gcaatagacc atgaacgcaa tcgacagagt gaagaaatcc agaggcatca
     acgagttagc ggagcagatc gaaccgctgg cccagagcat ggcgacactg gccgacgaag
     cccggcaggt catgagccag accaagcagg ccagcgaggc gcaggcggcg gagtggctga
     aagcccagcg ccagacaggg gcggcatggg tggagctggc caaagagttg cgggaggtag
     ccgccgaggt gagcagcgcc gcgcagagcg cccggagcgc gtcgcggggg tggcactgga
     agctatggct aaccgtgatg ctggcttcca tgatgcctac ggtggtgctg ctgatcgcat
     cgttgctctt gctcgacctg acgccactga caaccgagga cggctcgatc tggctgcgct
     tggtggcccg atgaagaacg acaggacttt gcaggccata ggccgacagc tcaaggccat
     gggctgtgag cgcttcgata tcggcgtcag ggacgcaccc accggccaga tgatgaaccg
     ggaatggtca gccgccgaag tgctccagaa cacgccatgg ctcaagcgga tgaatgccca
     gggcaatgac gtgtatatca ggcccgccga gcaggagcgg catggtctgg tgctggtgga
     cgacctcagc gagtttgacc tggatgacat gaaagccgag ggccgggagc ctgccctggt
     agtggaaacc agcccgaaga actatcaggc atgggtcaag gtggccgacg ccgcaggcgg
     tgaacttcgg gggcagattg cccggacgct ggccagcgag tacgacgccg acccggccag
     cgccgacagc cgccactatg gccgcttggc gggcttcacc aaccgcaagg acaagcacac
     cacccgcgcc ggttatcagc cgtgggtgct gctgcgtgaa tccaagggca agaccgccac
     cgctggcccg gcgctggtgc agcaggctgg ccagcagatc gagcaggccc agcggcagca
     ggagaaggcc cgcaggctgg ccagcctcga actgcccgag cggcagctta gccgccaccg
     gcgcacggcg ctggacgagt accgcagcga gatggccggg ctggtcaagc gcttcggtca
     tgacctcagc aagtgcgact ttatcgccgc gcagaagctg gccagccggg gccgcagtgc
     cgaggaaatc ggcaaggcca tggccgaggc cagcccagcg ctggcagagc gcaagcccgg
     ccacgaagcg gattacatcg agcgcaccgt cagcaaggtc atgggtctgc ccagcgtcca
     gcttgcgcgg gccgagctgg cacgggcacc ggcaccccgc cagcgaggca tggacagggg
     cgggccagat ttcagcatgt agtgcttgcg ttggtactca cgcctgttat actatgagta
     ctcacgcaca gaagggggtt ttatggaata cgaaaaaagc gcttcagggt cggtctacct
     gatcaaaagt gacaagggct attggttgcc cggtggcttt ggttatacgt caaacaaggc
     cgaggctggc cgcttttcag tcgctgatat ggccagcctt aaccttgacg gctgcacctt
     gtccttgttc cgcgaagaca agcctttcgg ccccggcaag tttctcggtg actgatatga
     aagaccaaaa ggacaagcag accggcgacc tgctggccag ccctgacgct gtacgccaag
     cgcgatatgc cgagcgcatg aaggccaaag ggatgcgtca gcgcaagttc tggctgaccg
     acgacgaata cgaggcgctg cgcgagtgcc tggaagaact cagagcggcg cagggcgggg
     gtagtgaccc cgccagcgcc taaccaccaa ctgcctgcaa aggaggcaat caatggctac
     ccataagcct atcaatattc tggaggcgtt cgcagcagcg ccgccaccgc tggactacgt
     tttgcccaac atggtggccg gtacggtcgg ggcgctggtg tcgcccggtg gtgccggtaa
     atccatgctg gccctgcaac tggccgcaca gattgcaggc gggccggatc tgctggaggt
     gggcgaactg cccaccggcc cggtgatcta cctgcccgcc gaagacccgc ccaccgccat
     tcatcaccgc ctgcacgccc ttggggcgca cctcagcgcc gaggaacggc aagccgtggc
     tgacggcctg ctgatccagc cgctgatcgg cagcctgccc aacatcatgg ccccggagtg
     gttcgacggc ctcaagcgcg ccgccgaggg ccgccgcctg atggtgctgg acacgctgcg
     ccggttccac atcgaggaag aaaacgccag cggccccatg gcccaggtca tcggtcgcat
     ggaggccatc gccgccgata ccgggtgctc tatcgtgttc ctgcaccatg ccagcaaggg
     cgcggccatg atgggcgcag gcgaccagca gcaggccagc cggggcagct cggtactggt
     cgataacatc cgctggcagt cctacctgtc gagcatgacc agcgccgagg ccgaggaatg
     gggtgtggac gacgaccagc gccggttctt cgtccgcttc ggtgtgagca aggccaacta
     tggcgcaccg ttcgctgatc ggtggttcag gcggcatgac ggcggggtgc tcaagcccgc
     cgtgctggag aggcagcgca agagcaaggg ggtgccccgt ggtgaagcct aagaacaagc
     acagcctcag ccacgtccgg cacgacccgg cgcactgtct ggcccccggc ctgttccgtg
     ccctcaagcg gggcgagcgc aagcgcagca agctggacgt gacgtatgac tacggcgacg
     gcaagcggat cgagttcagc ggcccggagc cgctgggcgc tgatgatctg cgcatcctgc
     aagggctggt ggccatggct gggcctaatg gcctagtgct tggcccggaa cccaagaccg
     aaggcggacg gcagctccgg ctgttcctgg aacccaagtg ggaggccgtc accgctgaat
     gccatgtggt caaaggtagc tatcgggcgc tggcaaagga aatcggggca gaggtcgata
     gtggtggggc gctcaagcac atacaggact gcatcgagcg cctttggaag gtatccatca
     tcgcccagaa tggccgcaag cggcaggggt ttcggctgct gtcggagtac gccagcgacg
     aggcggacgg gcgcctgtac gtggccctga accccttgat cgcgcaggcc gtcatgggtg
     gcggccagca tgtgcgcatc agcatggacg aggtgcgggc gctggacagc gaaaccgccc
     gcctgctgca ccagcggctg tgtggctgga tcgaccccgg caaaaccggc aaggcttcca
     tagatacctt gtgcggctat gtctggccgt cagaggccag tggttcgacc atgcgcaagc
     gccgcaagcg ggtgcgcgag gcgttgccgg agctggtcgc gctgggctgg acggtaaccg
     agttcgcggc gggcaagtac gacatcaccc ggcccaaggc ggcaggctga ccccccccac
     tctattgtaa acaagacatt tttatctttt atattcaatg gcttattttc ctgctaattg
     gtaataccat gaaaaatacc atgctcagaa aaggcttaac aatattttga aaaattgcct
     actgagcgct gccgcacagc tccataggcc gctttcctgg ctttgcttcc agatgtatgc
     tcttctgctc ccgaacgcca gcaagacgta gcccagcgcg tcggccagct tgcaattcgc
     gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     ccctgagaga gttgcagcaa gcggtccacg tggtttgccc cagcaggcga aaatcctgtt
     tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     gctatcatgc cataccgcga aaggttttgc accattcgat ggtgtcaacg taaatgccgc
     ttcgccttcg cgcgcgaatt gcaagctgat ccgggcttat cgactgcacg gtgcaccaat
     gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact
     gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat
     cataacggtt ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat
     aatgtgtgga attgtgagcg gataacaatt tcacacagga aacagaa
//