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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RL   Unpublished (1990).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
//