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ID   PATH3      preliminary; circular DNA; SYN; 3763 BP.
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AC   M33622; ATCC37697;
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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 pATH3 - 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   1-3763
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   pATH3
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RL   Unpublished (1990).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   The nucleotides remaining in the codon after cutting the vector with
CC   the given enzyme are: BamHI-1, ClaI-2, EcoRI-3, HindIII-1, SalI-1,
CC   XbaI-1.
CC   Restriction digests of the clone give the following sizes (kb):
CC   BamHI--3.8; EcoRI--3.8; HindIII--3.8. (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 (pATH3)
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 (pATH2)
CC   BR ()
CC   OF (pATH10)(pATH11)
CC   OR ()
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FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pATH1 remove EcoRI-BamHI 18bp 1406..1424,
FT                   \ MCS/3761bp
FT                   -> pATH3 3763bp"
FT   misc_binding    0..0
FT                   /note="MCS EcoRI-BamHI-XbaI-SalI-PstI-HindIII-ClaI,
FT                   from M13mp12"
FT   CDS             423..>1391
FT                   /note="GEN E. coli trpE' gene (for fusion)"
FT   CDS             0..0
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 3763 BP; 926 A; 942 C; 946 G; 949 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 ccccccgaat tcggggggat cctctagagt cgacctgcag cccaagctta
     tcgatgataa gctgtcaaac atgagaatta attcttgaag acgaaagggc ctcgtgatac
     gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc
     ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agg
//