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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RL Unpublished (1990).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
//