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ID   PATH2      preliminary; circular DNA; SYN; 3753 BP.
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AC   M33624; ATCC37696;
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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 pATH2 - 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   1-3753
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   pATH2
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RL   Unpublished (1990).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   The nucleotides remaining in the codon after cutting the vector with
CC   the given enzyme are: BamHI-3, ClaI-1, HindIII-3, SalI-3, SmaI-3,
CC   XbaI-3, XmaI-1.
CC   Restriction digests of the clone give the following sizes (kb):
CC   BamHI--3.75; HindIII--3.75. (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 (pATH2)
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)(pKRS101)(pBR322)
CC   BR (pATH series)
CC   OF (pATH3)(pATH10)(pATH11)
CC   OR ()
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FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pATH1 remove SmaI-BamHI 29bp 1395..1424,
FT                   \ MCS/3750bp
FT                   -> pATH2 3753bp"
FT   misc_binding    0..0
FT                   /note="MCS SmaI-BamHI-XbaI-SalI-PstI, from M13mp12"
FT   misc_binding    0..0
FT                   /note="SIT unique ClaI"
FT   misc_binding    0..0
FT                   /note="SIT unique HindIII"
FT   CDS             423..1454
FT                   /note="GEN E. coli trpE' gene (for fusion)"
FT   CDS             1670..2530
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 3753 BP; 924 A; 939 C; 943 G; 947 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 ccccggggat 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
//