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ID   PGEX1      preliminary; circular DNA; SYN; 4932 BP.
XX
AC   M21676; A01437; IG2001;
XX
DT   01-JUL-1993 (Rel. 7, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
XX
DE   E. coli plasmid vector pGEX-1 - complete.
XX
KW   cloning vector.
XX
OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
XX
RN   [1]
RC   plasmid from pSj1 & pIC19H
RC   pSj10 from plasmid & ptac or ptac12deltaEco
RC   p4.5 from pSj5 & pMC9
RC   pSj10Bam7Stop7 from pSj10
RC   pGEX-1 from pBR322 & pSj10Bam7Stop7 & p4.5 & oligo
RC   pGEX-2T from pGEX-1 & oligo, thrombin protease site
RC   pGEX-3X from pGEX-1 & oligo, factor Xa site
RA   Smith D.B., Johnson K.S.;
RT   "Single-step purification of polypeptides expressed in Escherichia
RT   coli as fusions with glutathione S-transferase";
RL   Gene 67:31-40(1988).
XX
RN   [2]
RC   pSj1 from ptac12 & Schistosoma japonicum Sj26
RC   pSj5 from ptac11 & oligo & pSj1, Schistosoma japonicum Sj26
RA   Smith D.B., Rubira M.R., Simpson R.J., Davern K.M., Tiu W.U.,
RA   Board P.G., Mitchell G.F.;
RT   "Expression of an enzymatically-active parasite molecule in
RT   Escherichia coli: Schistosoma japonicum glutathione S-transferase";
RL   Mol. Biochem. Parasitol. 27:249-256(1988).
XX
RN   [3]
RC   pMC1T63 from pMC1 & T63, lacIq gene/partial lacZ gene
RC   pMC9T63 from pMC1T63 & linker & pBR322
RC   pMC9 from pMC9T63
RC   pSVi2, pSVi4 from pML & SV40 ori & lacIq gene & partial lacZ gene
RC   pJYMib from pML & SV40 & lacIq gene & partial lacZ gene
RC   pSV2gptia, pSV2gptib from pSV2-gpt & SV40
RC   pdeltaH3ib from pSVi2
RA   Miller J.H., Lebkowski J.S., Greisen K.S., Calos M.P.;
RT   "Specificity of mutations in transfected DNA by mammalian cells";
RL   EMBO J. 3:3117-3121(1984).
XX
RN   [4]
RC   pJYMia, pJYMib from pML & SV40 & lacIq gene & partial lacZ gene
RC   pSV2gptia, pSV2gptib from pSV2-gpt & SV40
RC   pdeltaH3ia, pdeltaH3ib from pSVi2
RA   Lebkowski J.S., DuBridge R.B., Antell E.A., Greisen K.S., Calos M.P.;
RT   "Transfected DNA is mutated in monkey, mouse, and human cells";
RL   Mol. Cell. Biol. 4:1951-1960(1984).
XX
RN   [5]
RP   1-4932
RC   pGEX-1
RA   Davern K.M.;
RT   ;
RL   Unpublished (1991).
XX
CC   Expression vector permitting production of a fusion protein.
CC   (ATCC staff)
CC   The glutathione S-transferase (GST) fusion protein can be purified by
CC   glutathione affinity chromatography, and the desired polypeptide
CC   released from the fusion product by thrombin. [2]
CC   Constructed from pGEX-2T by inserting an oligonucleotide at the EcoRI
CC   site which encodes the glycine kinker and additional restriction
CC   sites to facilitate cloning in all reading frames. [2]
CC   The order of the major features in this plasmid are: Ptac - GST -
CC   thrombin cleavage site - BamHI site - SmaI site - glycine kinker -
CC   multiple cloning site - ampR - pBR322 ori - lacIq. [2]
CC   Restriction digests of the clone give the following sizes (kb):
CC   HindIII--5.0; BamHI--5.0; EcoRI--5.0; XbaI--5.0. (ATCC staff)
CC   Medium is 1227 LB plus ampicillin.
CC   NCBI gi: 208304
CC   NM (pGEX-1)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP (ATCC)
CC   HO (E.coli HB101)(E.coli)(E.coli TG1)(E.coli JM101)
CC   CP ()
CC   FN (cloning)
CC   SE ()
CC   PA ()
CC   BR ()
CC   OF ()
CC   OR ()
XX
FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. ptac12 EcoRI 2600bp, pBR322 4360..4360
FT                   2. Schistosoma japonicum EcoRI-EcoRI 780bp, Sj26
FT                   -> pSj1 3400bp
FT                   1. ptac12 EcoRI 2600bp, pBR322 4360..4360
FT                   fill in
FT                   -> ptac12deltaEco 2600bp
FT                   1. lambda
FT                   2. E. coli lac operon, lacI/lacPO/Z/Y/A
FT                   -> lambda h80dlac
FT                   1. pMB9 HpaI 5300bp
FT                   2. lambda h80dlac HindII-HindIII 2800bp, lacI/lacPO
FT                   -> pMC1 8100bp
FT                   1. E. coli, lacIq gene/partial lacZ gene
FT                   -> T63 [no HincII in lacI]
FT                   1. pMC1 8100bp, lacI gene
FT                   crossed with
FT                   2. T63, lacI gene with no HincII
FT                   -> pMC1T63
FT                   1. pMC1T63 HincII-HincII 1700bp, lacI to AA146 lacZ
FT                   EcoRI linker 6bp gaattc:EcoRI linker 6bp gaattc
FT                   2. pBR322 EcoRI 4361bp 4360..4360
FT                   -> pMC9T63 6100bp
FT                   1. pMC9T63 6100bp, lacI- gene
FT                   crossed with F'lac strain, lacI+ gene
FT                   -> pMC9 6100bp
FT                   1. pSj1 MnlI 3400bp,
FT                   \ Sj26 gene termination taa to tcggatcc
FT                   BamHI linker 6bp ggatcc
FT                   remove EcoRI-Sau96I, 5' Sj26 gene
FT                   BamHI-caccatgtcc-12..38 pSj1
FT                   BamHI-BamHI, Sj26 gene
FT                   2. pIC19H BamHI 2701bp 254..254
FT                   -> plasmid [unique SmaI-EcoRI-ClaI-EcoRV after Sj26]
FT                   1. plasmid BamHI-EcoRV, Sj26 gene/pIC19H 254..-..275
FT                   blunt end:blunt end
FT                   2. ptac12deltaEco PvuII 2600bp, pBR322 2069..2069
FT                   -> pSj10
FT                   1. ptac11 4600bp
FT                   2. oligo
FT                   3. pSj1 3400bp, Schistosoma japonicum Sj26
FT                   -> pSj5
FT                   1. pSj5 remove HindIII-NdeI, tet gene
FT                   Klenow:Klenow
FT                   2. pMC9 EcoRI-EcoRI 1700bp, lacIq gene/lacZ
FT                   Klenow:Klenow
FT                   -> p4.5
FT                   1. pSj10 BamHI, 5' Sj26 gene
FT                   Klenow
FT                   ClaI, 3' Sj26 gene
FT                   blunt end:blunt end
FT                   2. oligo 11bp tgactgactga
FT                   -> pSj10Bam7Stop7
FT                   1. p4.5 BclI-EcoRI, amp/ori/5' Sj26
FT                   :Klenow
FT                   2. pSj10Bam7Stop7 BclI-AccI, 3' Sj26/MCS/term/pBR322
FT                   :Klenow
FT                   -> plasmid
FT                   1. p4.5 EcoRI, 3' Sj26 gene
FT                   fill in
FT                   EcoRI, 5' Sj26 gene
FT                   mutagenesis to tac-gtattc-Sj26
FT                   -> plasmid2
FT                   1. plasmid2 BclI-BclI, 3' lacIq/tac/5' Sj26
FT                   2. plasmid BclI
FT                   -> pGEX-1 4932bp"
FT   promoter        0..0
FT                   /note="PRO E. coli tac promoter"
FT   CDS             258..944
FT                   /note="GEN Schistosoma japonicum
FT                   glutathione S-transferase (GST)"
FT   misc_binding    914..929
FT                   /note="MCS unique BamHI-SmaI-EcoRI"
FT   misc_binding    914..919
FT                   /note="SIT PstI"
FT   misc_binding    919..924
FT                   /note="SIT PstI"
FT   misc_binding    924..>929
FT                   /note="SIT PstI"
FT   misc_binding    914..929
FT                   /note="MCS BamHI-SmaI-EcoRI-XbaI-NcoI-SalI-
FT                   XhoI-SacI-HindIII"
FT   CDS             0..0
FT                   /note="ANT E. coli beta-lactamase gene (bla)
FT                   ampicillin resistance gene (apr/amp)"
FT   misc_binding    0..0
FT                   /note="SIT PstI"
FT   rep_origin      0..0
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322)"
FT   CDS             0..0
FT                   /note="REP E. coli lacIq repressor gene"
FT   misc_binding    0..0
FT                   /note="SIT EcoRV"
FT   CDS             0..0
FT                   /note="GEN E. coli beta-galactosidase gene (lacZ)"
XX
SQ   Sequence 4932 BP; 1222 A; 1190 C; 1278 G; 1242 T; 0 other;
     agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
     gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
     tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
     tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
     cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
     aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
     gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
     ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
     tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
     ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
     ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
     atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
     tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
     aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
     ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
     atcctccaaa atcggatccc cgggaattca tcgtgactga ctgacgatct gcctcgcgcg
     tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 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
     gctatgagaa 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 cgcataaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgtcaac
     caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg
     gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc
     gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtaacc
     tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg
     ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat
     ggcgttggaa tt
//