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ID   PEZZ18     preliminary; circular DNA; SYN; 4591 BP.
XX
AC   M74186; M18256;
XX
DT   02-NOV-1992 (Rel. 7, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
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DE   E. coli phagemid vector pEZZ18 - 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   1-4591
RC   pAZ from protein A signal & synthetic IgG-binding domain or Z domain
RC   pEZ from pAZ & pEMBL8+
RC   pEZZ from pEZ
RC   pEZZ8 from pEZZ & M13mp8
RC   pEZZ18 from pEZZ & M13mp18
RC   pEZZ5770 from pEZZ8 & oligo
RA   Loewenadler B., Jansson B., Paleus S., Holmgren E., Nilsson B.,
RA   Moks T., Palm G., Josephson S., Philipson L., Uhlen M.;
RT   "A gene fusion system for generating antibodies against short
RT   peptides";
RL   Gene 58:87-97(1987).
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RN   [2]
RP   1-4591
RC   from pRIT4
RC   M13Z from M13mp8 & Z domain
RC   pHL3 from pEMBL19 & pRIT4, protein A gene
RC   pHL3Z from pHL3 & M13Z
RC   pASZ from pHL3Z
RC   pEZ from pASZ & pEMBL8
RC   pEZZ from pEZ & one Z domain
RC   pEZV from pEZZ & three Z domains
RC   pEZX from pEZV & five Z domains
RA   Nilsson B., Moks T., Jansson B., Abrahmsen L., Elmblad A.,
RA   Holmgren E., Henrichson C., Jones T.A., Uhlen M.;
RT   "A synthetic IgG-binding domain based on staphylococcal protein A";
RL   Protein Engineering 1:107-113(1987).
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RN   [3]
RC   from pEZZ18
RA   Nilsson B., Forsberg G., Hartmanis M.;
RT   "Expression and purification of recombinant insulin-like growth
RT   factors from Escherichia coli";
RL   Meth. Enzymol. 198:3-16(1991).
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CC   The folding properties of the MW 14000 synthetic IgG binding domains
CC   do not interfere with the folding of the protein fused to them.
CC   NM (pEZZ18)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (phagemid)
CC   SP (Pharmacia)
CC   HO (E.coli JM105)
CC   CP ()
CC   FN (expression)
CC   SE (affinity)
CC   PA (pEMBL8+)(pAS)(pUC18)
CC   BR ()
CC   OF ()
CC   OR ()
XX
FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pRIT4 TaqI-EcoRI, protein A gene
FT                   2. pEMBL19+ HindIII 3960bp 232..232, MCS
FT                   fill in
FT                   remove EcoRI-ClaI 1062bp 283..1345, MCS
FT                   -> plasmid
FT                   1. plasmid TaqI/ClaI, pEMBL19+ 1345
FT                   fill in
FT                   NotI linker 10bp agcggccgct
FT                   -> pHL3
FT                   1. M13mp8 HindIII-EcoRI 7199bp 6262..7229..6232
FT                   2. oligo EcoRI-HindIII, Z fragment/IgG-binding domain
FT                   -> M13Z 7200bp
FT                   1. M13Z HindIII-EcoRI, Z fragment/IgG-binding domain
FT                   2. pHL3 remove small HindIII-EcoRI, protein A gene
FT                   -> pHL3Z
FT                   1. pHL3Z FspI
FT                   fill in
FT                   -> pASZ
FT                   1. pASZ TaqI, spa gene
FT                   NotI linker 10bp agcggccgct
FT                   NotI-BamHI
FT                   2. pEMBL8+ AccI 3939bp 246..246
FT                   Klenow
FT                   EcoRI 3939bp 229..229
FT                   NotI linker 10bp agcggccgct
FT                   remove small NotI-BamHI 10bp, pEMBL8+ 229..239/3929bp
FT                   -> pEZ [2 AccI around Z fragment]
FT                   1. pEZ AccI
FT                   2. pEZ AccI-AccI 174bp, IgG-binding domain or Z domain
FT                   -> pEZZ [2 Z fragments]
FT                   1. pEZZ remove EcoRI-HindIII
FT                   2. M13mp18 EcoRI-HindIII 51bp 6231..6282, MCS
FT                   -> pEZZ18 4591bp"
FT   misc_feature    1..2236
FT                   /note="from pEMBL8p"
FT   misc_recomb     2236..2237
FT                   /note="pEMBL8p DNA end/synthetic NotI linker
FT                   start"
FT   misc_feature    2237..2246
FT                   /note="SIT synthetic NotI linker"
FT   misc_binding    2238..2238
FT                   /note="SIT NotI"
FT   misc_recomb     2246..2247
FT                   /note="synthetic NotI linker end/Staphylococcus aureus
FT                   protein A promoter DNA start"
FT   promoter        2247..2431
FT                   /note="PRO S. aureus protein A signal sequence,
FT                   partial E domain, synthetic ZZ and E. coli lacZ' gene"
FT   CDS             2432..3157
FT                   /note="GEN S. aureus protein A signal sequence,
FT                   partial E domain, synthetic ZZ and E. coli lacZ' gene"
FT   misc_feature    2561..2734
FT                   /note="synthetic Z (domain 1)
FT                   (synthetic IgG binding domain)"
FT   misc_feature    2735..2908
FT                   /note="synthetic Z (domain 2)
FT                   (synthetic IgG binding domain)"
FT   promoter        0..0
FT                   /note="PRO E. coli lac gene"
FT   promoter        0..0
FT                   /note="PRO E. coli tac (trp -35 and lacUV5 -10)"
FT   promoter        0..0
FT                   /note="PRO E. coli spa gene"
FT   misc_binding    2912..2971
FT                   /note="MCS EcoRI-SacI-KpnI-SmaI-BamHI-XbaI-SalI-
FT                   BspMI-PstI-SphI-HindIII"
FT   CDS             0..0
FT                   /note="GEN E. coli beta-galactosidase gene (lacZ)
FT                   alpha peptide"
FT   sig_peptide     2432..2539
FT                   /note="S. aureus protein A signal sequence,
FT                   partial E domain, synthetic ZZ and lacZ'"
FT   misc_feature    2540..2560
FT                   /note="S. aureus E domain of protein A"
FT   mat_peptide     2540..3154
FT                   /note="S. aureus protein A signal sequence,
FT                   partial E domain, synthetic ZZ and E. coli lacZ' gene"
FT   misc_recomb     2560..2561
FT                   /note="Staphylococcus aureus partial E domain DNA
FT                   end/synthetic ZZ gene DNA start"
FT   misc_feature    2561..2908
FT                   /note="GEN S. aureus protein A analogue,
FT                   synthetic ZZ portion of gene"
FT   misc_feature    2561..2734
FT                   /note="synthetic ZZ"
FT   misc_feature    2735..2908
FT                   /note="synthetic ZZ"
FT   misc_recomb     2908..2909
FT                   /note="synthetic ZZ gene DNA end/synthetic linker end"
FT   misc_feature    2909..2913
FT                   /note="synthetic linker"
FT   misc_recomb     2913..2914
FT                   /note="synthetic linker end/pUC18 polylinker DNA
FT                   start"
FT   misc_feature    2914..2970
FT                   /note="MCS pUC18"
FT   misc_recomb     2970..2971
FT                   /note="pUC18 polylinker DNA end/pEMBL8p DNA start"
FT   misc_feature    2971..4591
FT                   /note="from pEMBL8p"
FT   misc_feature    2971..3157
FT                   /note="GEN E. coli beta-galactosidase gene (lacZ')
FT                   from pEMBL8p"
FT   rep_origin      0..0
FT                   /note="ORI bacteriophage f1 intergenic region"
FT   misc_binding    3982..3982
FT                   /note="SIT ClaI"
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)"
XX
SQ   Sequence 4591 BP; 1206 A; 1094 C; 1037 G; 1254 T; 0 other;
     gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     accatgatta cgaattagcg gccgctagcc gaaatagcgt gattttgcgg ttttaagcct
     tttacttcct gaataaatct ttcagcaaaa tatttatttt ataagttgta aaacttacct
     ttaaatttaa ttataaatat agattttagt attgcaatac ataattcgtt atattatgat
     gactttacaa atacatacag ggggtattaa tttgaaaaag aaaaacattt attcaattcg
     taaactaggt gtaggtattg catctgtaac tttaggtaca ttacttatat ctggtggcgt
     aacacctgct gcaaatgctg cgcaacacga tgaagccgta gacaacaaat tcaacaaaga
     acaacaaaac gcgttctatg agatcttaca tttacctaac ttaaacgaag aacaacgaaa
     cgccttcatc caaagtttaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc
     taaaaagcta aatgatgctc aggcgccgaa agtagacaac aaattcaaca aagaacaaca
     aaacgcgttc tatgagatct tacatttacc taacttaaac gaagaacaac gaaacgcctt
     catccaaagt ttaaaagatg acccaagcca aagcgctaac cttttagcag aagctaaaaa
     gctaaatgat gctcaggcgc cgaaagtaga cgcgaattcg agctcggtac ccggggatcc
     tctagagtcg acctgcaggc atgcaagctt ggcactggcc gtcgttttac aacgtcgtga
     ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     tggcgaatgg cgattccgtt gcaatggctg gcggtaatat tgttctggat attaccagca
     aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat caaagaagta
     ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc ctcactgatt
     ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct ttaatcggcc
     tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg ctcgtcaaag
     caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     caaaaattta acgcgaattt taacaaaata ttaacgttta caatttaaat atttgcttat
     acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat gattgacatg
     ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc aggcaatgac
     ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg aatttatcag
     ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt
     ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa
     atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg
     tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt
     ctttgccttg cctgtatgat ttattggatg ttggaattga tgcggtattt tctccttacg
     catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     aggttttcac cgtcatcacc gaaacgcgcg a
//