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ID PATH11 preliminary; circular DNA; SYN; 3772 BP.
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AC M33625; ATCC37699;
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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 pATH11 - 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 1-3772
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 pATH11
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 1-144
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RL Unpublished (1990).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 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 pATH11.
CC The nucleotides remaining in the codon after cutting the vector with
CC the given enzyme are: BamHI-1, ClaI-2, EcoRI-1, HindIII-1, PstI-2,
CC SacI-2, SalI-1, SmaI-1, XbaI-1, XmaI-2.
CC Restriction digests of the clone give the following sizes (kb):
CC BamHI--3.85; EcoRI--3.85; HindIII--3.85. (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 (pATH11)
CC CM (yes)
CC NA (ds-DNA)
CC TP (circular)
CC ST ()
CC TY (plasmid)
CC SP (ATCC)
CC HO (E.coli)
CC CP ()
CC FN (expression)(fusion)
CC SE ()
CC PA (pATH1)(pATH2)(pATH3)
CC BR (pATH10)
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 -> pATH11 3772bp [no PstI in amp]"
FT misc_binding 0..0
FT /note="MCS unique BamHI-SmaI-SstI-EcoRI, from M13mp12"
FT CDS 423..1487
FT /note="GEN E. coli trpE' gene (for fusion)"
FT CDS 1689..2549
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)"
FT misc_binding 0..0
FT /note="SIT unique ClaI"
FT misc_binding 0..0
FT /note="SIT unique HindIII"
FT misc_binding 0..0
FT /note="SIT unique PstI"
FT misc_binding 0..0
FT /note="SIT unique SacI"
FT misc_binding 0..0
FT /note="SIT unique SalI"
FT misc_binding 0..0
FT /note="SIT unique XbaI"
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SQ Sequence 3772 BP; 927 A; 946 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 ccccccggaa ttcgagctcg cccggggatc ctctagagtc gacctgcagc
ccaagcttat cgatgataag ctgtcaaaca tgagaattaa ttcttgaaga cgaaagggcc
tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag cattgagaaa
gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gg
//