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ID PEZZ18 preliminary; circular DNA; SYN; 4591 BP.
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AC M74186; M18256;
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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 ()
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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
//