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ID   PFXBLUE    preliminary; circular DNA; SYN; 3627 BP.
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AC   M36415; ATCC77333;
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DT   20-MAR-1991 (Rel. 6, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
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DE   E. coli plasmid vector pFXblue - 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   1-3627
RC   M13mp18-BamHI from M13mp18
RC   pfX8mcs from M13mp18-BamHI & pfX8
RC   pMb3-EcoRI from pMb3
RC   pc2fX from pMb3-EcoRI & pfX8mcs
RC   pfXblue from pc2fX & M13mp18
RA   Brandt M.E., Gabrik A.H., Vickery L.E.;
RT   "A vector for directional cloning and expression of polymerase chain
RT   reaction products in Escherichia coli";
RL   Gene 97:113-117(1991).
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RN   [2]
RC   pFXblue
RA   Vickery L.E.;
RT   ;
RL   Submitted (11-JUL-1990) to GenBank on computer-readable media by:
RL   Vickery L.E., Dept. of Physiology and Biophysics,
RL   Univ. of California, Irvine CA 92717.
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RN   [3]
RC   plasmid from pfX8 & human ferredoxin gene
RC   M13mp19::plasmid from M13mp19 & plasmid
RC   pHFdx1 from pMb3 & M13mp19::plasmid
RA   Coghlan V.M., Vickery L.E.;
RT   "Expresion of human ferredoxin and assembly of the [2Fe-2S] center
RT   in Escherichia coli";
RL   Proc. Natl. Acad. Sci. U.S.A. 86:835-839(1989).
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RN   [4]
RC   pfX8 from pUC8 & M13mp11 FX, factor Xa
RA   Glabe C.;
RT   ;
RL   Unpublished (1989).
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RN   [5]
RC   pMb3 from lambda, cII gene
RA   Varadarajan R., Szabo A., Boxer S.G.;
RT   "Cloning, expression in Escherichia coli, and reconstitution of
RT   human myoglobin";
RL   Proc. Natl. Acad. Sci. U.S.A. 82:5681-5684(1985).
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CC   Deposited by: Mark E. Brandt
CC   Restriction digests of the plasmid give the following sizes (kb):
CC   BamHI--3.574, 0.053; BglI--1.843, 1.784; PstI--2.072, 1.555;
CC   PvuI--2.063, 1.564; PvuII--2.972, 0.655; SacI--3.257, 0.37.
CC   (GenBank/EMBL record)
CC   Expression vector permitting the production of a fusion protein that
CC   can be trimmed by cleavage with the coagulation factor Xa protease.
CC   A modified lacZ' allows blue/white screening of colonies. [1]
CC   Especially useful for the expression of PCR products. [1]
CC   Inserts can be cloned using the 5' StuI site and one other MCS
CC   restriction site matching the 3' end of the insert.  The vector
CC   supplies the AUG initiator codon. (personal communication)
CC   The insert to be cloned should have a phosphorylated 5' blunt end with
CC   the first base corresponding to the first base of the first codon.
CC   (personal communication)
CC   The order of the major features of the plasmid is:  pMB1 ori - ampR -
CC   lambdaPL promoter - lambda cII - factor Xa recognition sequence -
CC   StuI/MCS/HindIII/modified lacZ'. [1]
CC   Restriction digests of the clone give the following sizes (kb):
CC   BglI--1.8 (doublet); EcoRI--3.6; PstI--2.1, 1.55. (ATCC staff)
CC   Medium is 1227 LB plus ampicillin.
CC   NM (pFXblue)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP ()
CC   HO (E.coli)(E.coli HB101)
CC   CP ()
CC   FN (expression)(fusion)(cloning directional)
CC   SE (color blue/white)
CC   PA (lambda)(pBR322)
CC   BR ()
CC   OF (pcIIhFR also from pUC/AR)(phFR also from pdeltablue)
CC   OF (phFR18+ from pUC/AR18+)
CC   OR ()
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FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. lambda, cII gene
FT                   -> pMb3
FT                   1. pUC8 2665bp
FT                   2. M13mp11 FX, factor Xa
FT                   -> pfX8 2700bp
FT                   1. M13mp18 BamHI 7249bp 6252..6252, MCS
FT                   mung bean nuclease
FT                   -> M13mp18-BamHI 7200bp
FT                   1. M13mp18-BamHI BglII-EcoRI 700bp, MCS
FT                   2. linker StuI-EcoRI 15bp cctccgcggccatgg
FT                   -> plasmid [StuI-EcoRI-HindIII-BglII]
FT                   1. plasmid StuI-HindIII 62bp, MCS
FT                   2. pfX8 remove small StuI-HindIII, factor Xa
FT                   -> pfX8mcs 2700bp [factor Xa/linker/MCS]
FT                   1. pMb3 EcoRI, MCS
FT                   mung bean nuclease
FT                   -> pMb3-EcoRI
FT                   1. pMb3-EcoRI remove HindIII-BamHI, 3' to lambda cII
FT                   2. pfX8mcs small HindIII-BamHI 79bp, Xa/linker/MCS
FT                   -> pc2fX [lambda cII/factor Xa/linker/MCS]
FT                   1. pc2fX HindIII
FT                   mung bean nuclease
FT                   StuI
FT                   calf intestinal alkaline phosphatase
FT                   2. M13mp18
FT                   PCR, lacZ gene modified
FT                   -> pfXblue 3627bp [lambda cII/factor Xa/lacZ mod]"
FT   promoter        0..0
FT                   /note="PRO bacteriophage lambda PL"
FT   CDS             826..918
FT                   /note="GEN bacteriophage lambda cII gene"
FT   CDS             826..1446
FT                   /note="GEN E. coli pFXblue fusion protein"
FT   misc_recomb     918..919
FT                   /note="lambda cII peptide DNA end /
FT                   factor Xa linker peptide DNA start"
FT   misc_recomb     939..940
FT                   /note="factor Xa linker peptide DNA end/modified lacZ'
FT                   gene DNA start"
FT   misc_binding    0..0
FT                   /note="MCS StuI-NcoI-StyI-EcoRI-Asp718-KpnI-SmaI-
FT                   XmaI-XbaI-AccI-SalI-BspMI-SphI-HindIII"
FT   misc_binding    0..0
FT                   /note="SIT unique StuI-NcoI-EcoRI-Asp718-KpnI-SmaI-
FT                   XmaI-XbaI-AccI-SalI-SphI-HindIII"
FT   CDS             940..1446
FT                   /note="GEN E. coli beta-galactosidase gene (lacZ)
FT                   alpha peptide"
FT   CDS             0..0
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 3627 BP; 956 A; 911 C; 860 G; 900 T; 0 other;
     attatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg
     aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc
     aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc cctcacttct
     ggctggatga tggggcgatt caggctggta tgagtcagca acaccttctt cacgaggcag
     acctcagcgc cggtgatgcc gacgatgcgt ccggcgtaga ggatctctca cctaccaaac
     aatgcccccc tgcaaaaaat aaattcatat aaaaaacata cagataacca tctgcggtga
     taaattatct ctggcggtgt tgacataaat accactggcg gtgatactga gcacatcagg
     caggacgcac tgaccaccat gaaggtgacg ctcttaaaat taagccctga agaagggcag
     cattcaaagc agaaggcttt ggggtgtgtg atacgaaacg aagcattggc gagctcgccc
     ctaaagatca acaaggccga aagatttttt taactataaa cgctgatgga ggcgtttatg
     cccaagaggt aaagcccttc ccgagtaaca aaaaaacaac agcataaata accccgctct
     tacacattcc agccctgaaa aagggcatca aattaaacca cacctatggt gtatgcattt
     atttgcatac attcaatcaa ttgttatcta aggaaatact tacatatggt tcgtgcaaac
     aaacgcaacg aggctctacg aatcgagagt gcgttgctta acaaaatcgc aatgcttgga
     actgagaaga cagcggaagg gggatccatc gagggtaggc ctaccatggt tacgaattcg
     agctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagctt ggcactggcc
     gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
     gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atacggtcgt cgtcccctca
     aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta tcccattacg
     gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat
     gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttcctatt
     ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     ttacaattta aatatttgct tatacaatct tcctgttttt ggggcttttc tgattatcaa
     ccgcggtaca tatgatggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     gaatactcat actcttcctt tttcaat
//