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ID   PATH10     preliminary; circular DNA; SYN; 3771 BP.
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AC   M33623; ATCC37698;
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DT   28-JUL-1990 (Rel. 5, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
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DE   E. coli plasmid vector pATH10 - complete.
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KW   cloning vector.
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OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
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RN   [1]
RP   1-3771
RC   pATH1 from pKRS101 & linker
RC   pATH2 from pATH1 & linker
RC   pATH3 from pATH1 & linker
RC   pATH10 from pATH1 & pUC8
RC   pATH11 from pATH1 & pUC8
RC   pATH20 from pATH1 & pUC18
RC   pATH21 from pATH1 & pUC18
RC   pATH22 from pATH1 & pUC18
RC   pATH23 from pATH1 & pUC18
RA   Koerner T.J., Hill J.E., Myers A.M., Tzagoloff A.;
RT   "High-expression vectors with multiple cloning sites for construction
RT   of trpE-fusion genes: pATH vectors";
RL   Meth. Enzymol. 194:477-490(1991).
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RN   [2]
RC   pATH10
RA   Hill J.E.;
RT   ;
RL   Submitted (06-APR-1990) to GenBank on computer-readable media by:
RL   Hill J.E., .
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RN   [3]
RP   1-147
RC   pATH2 from pATH1 & linker
RC   pATH3 from pATH1 & linker
RC   pATH10 from pATH1 & pUC8
RC   pATH11 from pATH1 & pUC8
RA   Gunsalus R.P.;
RT   ;
RL   Unpublished (1990).
RL   Gunsalus R.P., Dept. of Microbiology, UCLA, Los Angeles CA.
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CC   The PvuII-HindIII fragment from the 5' end of the trp operon
CC   through nt 1999 of ECOTGP, which is in trpD CDS, was ligated to
CC   the HindIII-PvuII fragment of pBR322 containing the bla gene
CC   for Amp-resistance and origin of replication, but not the rop gene,
CC   which encodes a negative regulator of ColE1 replication.
CC   In addition, the EcoRI site in the pBR322 backbone was eliminated
CC   to make plasmid pKRS101.
CC   The BglII-HindIII fragment (nt 1392 of trpE to the end of the trpD
CC   sequence present in pKRS101) was replaced with a BamHI-EcoRI
CC   fragment and an EcoRI-HindIII fragment, both from the MCS of
CC   M13mp12 to make plasmid pATH1 (see GenBank M32985).
CC   The SmaI-SmaI fragment from the MCS of pATH1 was deleted and the
CC   remaining plasmid religated to make plasmid pATH2 (see
CC   GenBank M33624).
CC   An EcoRI linker was inserted at the remaining SmaI site of pATH2
CC   replacing the SmaI site and changing the reading frames of the
CC   other sites in the MCS to make pATH3.
CC   An interim vector was constructed by inserting an EcoRI linker
CC   at the remaining SmaI site of pATH2.
CC   The EcoRI-HindIII fragment of MCS in this interim vector was replaced
CC   with the EcoRI-HindIII fragment containing the MCS of M13mp12.
CC   Tha AvaII-AvaII fragment that spanned the PstI site in
CC   the bla gene of this interim vector was replaced with the
CC   corresponding AvaII fragment from pUC8, eliminating this PstI site,
CC   making the PstI site in the MCS unique, to form plasmid pATH10.
CC   The nucleotides remaining in the codon after cutting the vector with
CC   the given enzyme are: BamHI-3, ClaI-1, EcoRI-3, HindIII-3, PstI-1,
CC   SacI-1, SalI-3, SmaI-3, XbaI-3, XmaI-1.
CC   Restriction digests of the clone give the following sizes (kb):
CC   BamHI--4.0; EcoRI--4.0; HindIII--4.0. (ATCC staff)
CC   Nucleotide 1 is the first in the trp fragment, at the beginning of a
CC   PvuII site 261 nucleotides upstream of the transcription start site.
CC   The multiple cloning site follows codon 323 of trpE.
CC   To prevent recircularization of the vector, the depositors recommend
CC   including 30 - 50 U of calf alkaline phosphatase in the restriction
CC   enzyme digestion used to prepare the vector.
CC   Escherichia coli containing pATH vectors should be grown on medium
CC   supplemented with tryptophan to prevent expression. Storage as DNA at
CC   -20C is preferred; long term storage as cells may result in selection
CC   of non-expressing promoter mutations.
CC   Fusion proteins are produced in Escherichia coli usually in an
CC   insoluble form which facilitates purification. Hybrid proteins larger
CC   than 90 kDa are not expressed as efficiently as shorter ones.
CC   This is one of a series of vectors (ATCC 37695-37703) designed to
CC   facilitate expression of an open reading frame as a trpE fusion
CC   protein.
CC   Medium is -1 1065 plus ampicillin (50 ug/ml) & tryptophan (10 ug/ml).
CC   NM (pATH10)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP (ATCC)
CC   HO (E.coli RR1)(E.coli)
CC   CP ()
CC   FN (expression)(fusion)
CC   SE ()
CC   PA (pATH1)(pATH2)(pATH3)
CC   BR (pATH11)
CC   OF ()
CC   OR ()
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FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pATH1 remove SmaI-EcoRI 11bp 1395..1406,
FT                   \ MCS/3768bp
FT                   remove AvaII-AvaII 222bp 2120..2342, amp gene/3546bp
FT                   2. pUC8 AvaII-AvaII 222bp 1288..1510, amp gene
FT                   -> pATH10 3771bp [no PstI in amp]"
FT   misc_binding    0..0
FT                   /note="MCS unique EcoRI-SacI-SmaI-BamHI-XbaI-SalI-
FT                   PstI-HindIII-ClaI, from M13mp12"
FT   CDS             423..1472
FT                   /note="GEN E. coli trpE' gene (for fusion)"
FT   CDS             1688..2548
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp)"
FT   rep_origin      0..0
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322)"
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SQ   Sequence 3771 BP; 927 A; 945 C; 948 G; 951 T; 0 other;
     cagctgtggt gtcatggtcg gtgatcgcta gggtgccgag cgcatctcga ctgcacggtg
     caccaatgct tctggcgtca ggtagttatt ggaaagctgt ggtatggctg tgcaggtcgt
     aaatcactgc ataactcgct gctgcctaag gcgcactccc gttctggata atgttttttg
     cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     gaactagtta actagtacgc aagttcacgt aaaaagggta tcgacaatga aagcaatttt
     cgtactgaaa ggttggtggc gcacttcctg aaacgggcag tgtattcacc atgcgtaaag
     caatcagata cccagcccgc ctaatgagcg ggcttttttt tgaacaaaat tagagaataa
     caatgcaaac acaaaaaccg actctcgaac tgctaacctg cgaaggcgct tatcgcgaca
     atcccaccgc gctttttcac cagttgtgtg gggatcgtcc ggcaacgctg ctgctggaat
     ccgcagatat cgacagcaaa gatgatttaa aaagcctgct gctggtagac agtgcgctgc
     gcattacagc tttaggtgac actgtcacaa tccaggcact ttccggcaac ggcgaagccc
     tcctggcact actggataac gccctgcctg cgggtgtgga aagtgaacaa tcaccaaact
     gccgtgtgct gcgcttcccc cctgtcagtc cactgctgga tgaagacgcc cgcttatgct
     ccctttcggt ttttgacgct ttccgtttat tgcagaatct gttgaatgta ccgaaggaag
     aacgagaagc catgttcttc agcggcctgt tctcttatga ccttgtggcg ggatttgaag
     atttaccgca actgtcagcg gaaaataact gccctgattt ctgtttttat ctcgctgaaa
     cgctgatggt gattgaccat cagaaaaaaa gcacccgtat tcaggccagc ctgtttgctc
     cgaatgaaga agaaaaacaa cgtctcactg ctcgcctgaa cgaactacgt cagcaactga
     ccgaagccgc gccgccgctg ccagtggttt ccgtgccgca tatgcgttgt gaatgtaatc
     agagcgatga agagttcggt ggcgtagtgc gtttgttgca aaaagcgatt cgcgctggag
     aaattttcca ggtggtgcca tctcgccgtt tctctctgcc ctgcccgtca ccgctggcgg
     cctattacgt gctgaaaaag agtaatccca gcccgtacat gttttttatg caggataatg
     atttcaccct atttggcgcg tcgccggaaa gctcgctcaa gtatgatgcc accagccgcc
     agattgagat cccccggaat tcgagctcgc ccggggatcc tctagagtcg acctgcagcc
     caagcttatc gatgataagc tgtcaaacat gagaattaat tcttgaagac gaaagggcct
     cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag
     cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag g
//