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ID   PNP6CRMCH  preliminary; circular DNA; SYN; 4477 BP.
XX
AC   M88535;
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DT   25-SEP-1992 (Rel. 7, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
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DE   E. coli plasmid vector pNP6CRMCTAP-Hir - 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   1-4477
RC   pNP6dRI from pNP6
RC   pNP6CTAP, pNP6CRM-CTAP, pNP6COL-CTAP, pBR-CRM-CTAP from pNP6dRI
RA   Waleh N.S., Sohel I., Lazar J.B., Hudson D.V., Sze P., Underhill P.,
RA   Johnson P.H.;
RT   "Expression of synthetic genes encoding fused proteins under tight
RT   control of modified regulatory regions of the colicin operon";
RL   Gene 117:7-14(1992).
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CC   NM (pNP6CRMCTAP-Hir)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP ()
CC   HO (E.coli)
CC   CP ()
CC   FN (cloning)(expression)
CC   SE ()
CC   PA ()
CC   BR ()
CC   OF ()
CC   OR ()
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FH   Key             Location/Qualifiers
FH
FT   misc_feature    0..0
FT                   /note="1. pBR-CRM-CTAP
FT                   -> plasmid
FT                   1. plasmid EcoRI, 3' to CTAP gene
FT                   2. oligo EcoRI-EcoRI 230bp, hir gene
FT                   -> pNP6CRM-CTAP-Hir 4477bp"
FT   -35_signal      3560..3565
FT                   /note="S35 E. coli colicin E1 gene"
FT   -10_signal      3583..3588
FT                   /note="S10 E. coli colicin E1 gene"
FT   promoter        3595..3627
FT                   /note="PRO E. coli colicin E1 gene"
FT   RBS             3661..3665
FT                   /note="RBS E. coli colicin E1 gene"
FT   sig_peptide     3673..3690
FT                   /note="leader peptide for CTAP-III(Leu21)-hirudin
FT                   fusion protein"
FT   mat_peptide     3691..3945
FT                   /note="E. coli connective tissue activating peptide
FT                   III (Leu21 or CTAP-III); putative"
FT   misc_recomb     3945..3946
FT                   /note="junction between CTAP-III (Leu21) and linker
FT                   sequence"
FT   misc_feature    3946..3963
FT                   /note="linker sequence between CTAP-III (Leu21) and
FT                   hirudin"
FT   misc_recomb     3963..3964
FT                   /note="junction between linker sequence and hirudin"
FT   mat_peptide     3964..4158
FT                   /note="vertebrate hirudin mature peptide (inhibits
FT                   alpha-thrombin); putative;
FT                   hirudin can be released from the CTAP-III-hirudin
FT                   fusion by cleavage with CNBr"
FT   terminator      4412..4442
FT                   /note="TER E. coli Rho-dependent terminator; putative"
FT   CDS             3673..4161
FT                   /note="GEN E. coli connective tissue activating
FT                   peptide III (Leu21 or CTAP-III) and hirudin fusion
FT                   protein"
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SQ   Sequence 4477 BP; 1099 A; 1177 C; 1117 G; 1084 T; 0 other;
     ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtcgacg
     cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc
     gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga
     tcgctcgcgg ctcttaccag cctaacttcg atcactggac cgctgatcgt cacggcgatt
     tatgccgcct cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac
     cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg
     gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct
     tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca
     tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca
     tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     ccatgaacag aaattccccc ttacacggag gcatcaagtg accaaacagg aaaaaaccgc
     ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     ccacctgacg tcgacagtct tgacagggaa aatgcagcgg cgtataatgt gtgctgtata
     taaaaccagt ggttatatgt acagtattta tttgttaact cgagtgtttt aaaagtcaaa
     gaggatttta taatggaaac cgcggtaatg aacctggcta aaggtaaaga agaaagcttg
     gactctgact tatacgctga gctccgttgc ctgtgcatca aaactacttc tgggatccac
     ccgaaaaaca tccagtctct ggaagttatc ggtaaaggca ctcactgcaa ccaggttgaa
     gttatcgcta ctctgaaaaa cggtcgtaaa atctgtctag acccggacgc tccacgtatc
     aagaagatcg ttcagaaaaa actggctggt gacgaatctg ctgacagaat tctcgttaac
     atggttgtat acactgattg caccgaatct ggtcagaacc tgtgcctgtg tgaaggttcc
     aacgtatgcg gccaaggtaa caagtgcatc ctgggatccg acggcgagaa gaaccagtgc
     gtaaccggtg aaggtacccc gaaaccgcag tctcacaacg acggcgactt cgaagagatc
     ccggaggaat acctgcaata atgactgcag aagcttgaat tcgtatgccg tgcccggatg
     gtccggactc cggccgtcag ttcgctcgtt cttgctacca ggacccgtta ccctgcagct
     ggcttctgtt tgcgatccag gctacatcgg ttctcgttgc gacgactaat gactgcagaa
     gcttcaattc tatgctccta tattgataag aataaactta atactataaa tgaggtgtta
     gggatttaat tattctttat tgatataaaa agtcctagca atccaaatgg gattgctagg
     accaaacccc cccccccccc cccccccccc ccctgca
//