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ID   PATH1      preliminary; circular DNA; SYN; 3779 BP.
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AC   M32985; ATCC37695;
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DT   23-MAY-1990 (Rel. 5, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
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DE   E. coli plasmid vector pATH1 - 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   1-3779
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]
RP   1-3779
RC   pATH1
RA   Tzagoloff A.;
RT   ;
RL   Submitted (18-MAR-1990) to GenBank on computer-readable media by:
RL   Tzagoloff A., Biol. Dept., Columbia Univ., NY, NY 10027.
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RN   [3]
RC   BE5 from adenovirus 2 E1A
RC   C131 from adenovirus 2
RC   pKRS101 from pKJB1 & pPS21, E. coli trpE gene
RC   phage from M13mp9 & BE5, adenovirus 2 E1A
RC   pKRS102 from pKRS101 & phage
RC   pKRS103 from pKRS102 & C131, adenovirus 2
RC   plasmid from pUC8CM & adenovirus 5 E1B
RC   pKRS107 from pKRS101 & plasmid
RC   pKJB231.1 from pBR322 & phage
RC   phage2 from M13mp11 & adenovirus 5 E1B
RC   pDR21 from pKJB231.1 & phage2
RA   Spindler K.R., Rosser D.S., Berk A.J.;
RT   "Analysis of adenovirus transforming proteins from early regions
RT   1A and 1B with antisera to inducible fusion antigens produced in
RT   Escherichia coli";
RL   J. Virol. 49:132-141(1984).
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RN   [4]
RC   pPS21 from E. coli trpE gene
RA   Gunsalus R.P.;
RT   ;
RL   Unpublished (1984).
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RN   [5]
RC   pUC8CM from pUC8 & cat gene
RC   pKJB1 from pUC8CM
RC   pKJB231.1 from pKJB1
RA   Buckley K.;
RT   ;
RL   Unpublished (1984).
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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 nucleotides remaining in the codon after cutting the vector with
CC   the given enzyme are (S-not for 5'-juncture because of stop codon in
CC   frame): BamHI-2, ClaI-3S, EcoRI-2, HindIII-2S, SalI-2S, XbaI-2S.
CC   Restriction digests of the clone give the following sizes (kb):
CC   BamHI--3.8; EcoRI--3.8; HindIII--3.8. (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   pATH1 was constructed from the following: 1-1391, the trp promoter
CC   and most of trpE; 1392-1455, multiple cloning sequence; 1456-3779,
CC   ClaI/PvuII fragment of pBR322, including bla (1696-2556) and ori.
CC   The EcoRI site was altered.
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   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. [1]
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 (pATH1)
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 (pKRS101)(pBR322)(M13mp12)
CC   BR ()
CC   OF (pATH2)(pATH3)(pATH10)(pATH11)
CC   OR ()
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FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pUC8 2665bp, TaqI = 248 998 2442
FT                   2. Tn9 TaqI-TaqI 773bp 215..988, cat gene/#V00622
FT                   -> pUC8CM 3438bp
FT                   1. pUC8CM EcoRI 3438bp 231..231
FT                   fill in
FT                   -> pKJB1 3446bp [no EcoRI]
FT                   1. E. coli trpE operon
FT                   -> pPS21
FT                   1. pKJB1 PvuII-HindIII 3351bp 356..-..261, ori/amp
FT                   \ may be pUC8 261..2665..55
FT                   2. pPS21 PvuII-HindIII 1391bp, E. coli trpE -260/+1999
FT                   -> plasmid 4742bp
FT                   1. plasmid EcoRI 4742bp, E. coli trpE -260/+1999
FT                   fill in
FT                   -> pKRS101 4750bp
FT                   1. pKRS101 remove BglII-HindIII, trpE 1392 to trpD end
FT                   \ 3700bp
FT                   2. M13mp12 EcoRI-BamHI 18bp, MCS/M13mp10 6232..6250
FT                   3. M13mp12 EcoRI-HindIII 45bp, MCS/M13mp10 6232..6277
FT                   -> pATH1 3779bp"
FT   rep_origin      0..0
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322)"
FT   CDS             0..0
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp)"
FT   CDS             423..1433
FT                   /note="GEN E. coli trpE gene (for fusion)"
FT   misc_binding    1392..1455
FT                   /note="MCS SmaI-SacI-EcoRI-SacI-SmaI-BamHI-XbaI-
FT                   SalI-PstI-HindIII-ClaI, from M13mp12"
FT   misc_binding    1392..1455
FT                   /note="MCS unique EcoRI-BamHI-XbaI-SalI-HindIII"
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SQ   Sequence 3779 BP; 928 A; 948 C; 952 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 ccccgggcga gctcgaattc gagctcgccc ggggatcctc tagagtcgac
     ctgcagccca agcttatcga tgataagctg tcaaacatga gaattaattc ttgaagacga
     aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcat
     tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagg
//