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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RL Unpublished (1990).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
//