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ID   PJIR418    preliminary; circular DNA; SYN; 7358 BP.
AC   M77169; ATCC77387;
DT   15-SEP-1992 (Rel. 7, Created)
DT   01-JUL-1995 (Rel. 12, Last updated, Version 1)
DE   C. perfringens/E. coli plasmid vector pJIR418 - complete.
KW   cloning vector.
OS   Cloning vector
OC   Artificial sequences; Cloning vehicles.
RN   [1]
RP   1-7358
RC   pJIR145 from pJIR71 & Tn1725, tet gene
RC   pJIR274 from pUC18 & pJIR62, C.perfringens cat gene
RC   pJIR275 from pJIR274 & pJIR71, C.perfringens tet gene
RC   pJIR282 from pJIR145 & pJIR275
RC   pJIR292 from pJIR229 & pJIR275
RC   pJIR297 from pJIR292 & pIP404
RC   pJIR335 from pJIR297
RC   pJIR403 from pJIR335
RC   pJIR408 from pJIR403
RC   pJIR415 from pJIR408
RC   pJIR418 from pJIR415 & pUC18
RA   Sloan J., Warner T.A., Scott P.T., Bannam T.L., Berryman D.I.,
RA   Rood J.I.;
RT   "Construction of a sequenced Clostridium perfringens - Escherichia
RT   coli shuttle plasmid";
RL   Plasmid 27:207-219(1992).
RN   [2]
RC   [pIP401 from C.perfringens]
RC   pIP404 from C.perfringens
RC   pTG15 from pUC8 & pIP404
RC   pTG16 from pUC8 & pIP404
RC   pTG67 from pUC18 & pIP404
RA   Garnier T., Cole S.T.;
RT   "Identification and molecular genetic analysis of replication
RT   functions of the bacteriocinogenic plasmid pIP404 from
RT   Clostridium perfringens";
RL   Plasmid 19:134-150(1988).
RN   [3]
RC   pJIR418 from pJIR415 & pUC18
RA   Bannam T.L., Rood J.I.;
RT   "Clostridium perfringens-Escherichia coli shuttle vectors that
RT   carry single antibiotic resistance determinants";
RL   Plasmid 29:233-235(1993).
RN   [4]
RC   pJIR190, pJIR122 from pUC18 & C.perfringens ermP gene
RC   pJIR155 from pJIR122
RC   pJIR208 from pJIR122
RC   pJIR211, pJIR212 from pJIR208
RC   pJIR229, pJIR231 from pJIR208
RC   pJIR233 from pJIR231
RA   Berryman D.I., Rood J.I.;
RT   "Cloning and hybridization analysis of ermP, a
RT   macrolide-lincosamide-streptogramin B resistance determinant
RT   from Clostridium perfringens";
RL   Antimicrob. Agents Chemother. 33:1346-1353(1989).
RN   [5]
RC   pJU121 from C.perfringens
RC   pJU122 from C.perfringens
RC   pJU123 from C.perfringens
RC   pJU124 from C.perfringens
RC   pJU281 from C.perfringens
RC   pJU282 from C.perfringens
RC   pJU283 from C.perfringens
RC   pJU284 from C.perfringens
RC   pJU1 from pBR322 & pJU281
RC   pJU3 from pBR322 & pJU121
RC   pJU5 from pBR322 & pJU122
RC   pJU7 from pJU1 & pCW3
RC   pJU10 from pJU1 & pJU124
RC   pJU12 from pJU3 & pJU7
RC   pJU13 from pJU5 & pJU7
RC   pJU16 from pJU5 & pJU10
RA   Squires C.H., Heffner D.L., Evans R.J., Kopp B.J., Yarus M.J.;
RT   "Shuttle plasmids for Escherichia coli and Clostridium perfringens";
RL   J. Bacteriol. 159:465-471(1984).
RN   [6]
RC   pHR100 from pSL100 & pJU122, C.perfringens ori
RC   pHR104 from pHR100 & pJIR62
RC   pHR106 from pHR104
RA   Roberts L., Holmes W.M., Hylemon P.B.;
RT   "Development of a new shuttle plasmid system for Escherichia coli
RT   and Clostridium perfringens";
RL   Appl. Environ. Microbiol. 54:268-270(1988).
RN   [7]
RC   pHB101 from C.perfringens
RC   pAK101 from pBR322 & pHB101
RC   pAK201 from pAK101 & pHR106, C.perfringens cat gene
RA   Kim A.Y., Blaschek H.P.;
RT   "Construction of an Escherichia coli-Clostridium perfringens shuttle
RT   vector and plasmid transformation of Clostridium perfringens";
RL   Appl. Environ. Microbiol. 55:360-365(1989).
RN   [8]
RC   pSB92 from pHG165 & pCP1
RC   pSB92A2 from pSB92 & pJIR62, C.perfringens cat gene
RC   pSB134 from pSB92A2
RA   Phillips-Jones M.K.;
RT   "Plasmid transformation of Clostridium perfringens by
RT   electroporation methods";
RL   FEMS Microbiol. Lett. 66:221-226(1990).
RN   [9]
RC   pJIR2 [similar to pCW3] from C.perfringens
RC   pJIR4 [similar to pCW3] from C.perfringens
RC   pJIR19 from C.perfringens
RC   pJIR20 from C.perfringens
RC   pJIR21 from C.perfringens
RC   pJIR22 from C.perfringens
RC   pJIR23 from pIP401
RC   pJIR24 from pJIR25
RC   pJIR25 from C.perfringens
RC   pJIR26 from pJIR27
RC   pJIR27 from C.perfringens
RC   pJIR28 from pJIR29
RC   pJIR29 from C.perfringens
RC   pJIR30 from C.perfringens
RC   pJIR64 from C.perfringens
RC   pJIR45 from pUC18 & pIP401
RC   pJIR47 from pUC18 & pJIR23
RC   pJIR52 from pUC18 & pJIR45
RC   pJIR62 from pUC18 & pJIR52
RA   Abraham L.J., Wales A.J., Rood J.I.;
RT   "Worldwide distribution of the conjugative Clostridium perfringens
RT   tetracycline resistance plasmid, pCW3";
RL   Plasmid 14:37-46(1985).
RN   [10]
RC   pJIR71 from pJIR39 & pUC18
RC   pJIR144, pJIR145, pJIR146, pJIR147 from pJIR71 & Tn1725
RC   pJIR148, pJIR149 from pJIR71 & Tn1725
RA   Abraham L.J., Berryman D.I., Rood J.I.;
RT   "Hybridization analysis of the class P tetracycline resistance
RT   determinant from the Clostridium perfringens R-plasmid pCW3";
RL   Plasmid 19:113-120(1988).
RN   [11]
RC   phage from lambda & C.perfringens alpha toxin gene
RC   pTOX5, pTOX6 from pTZ18R & phage, C.perfringens phospholipase C gene
RA   Saint-Joanis B., Garnier T., Cole S.T.;
RT   "Gene clonign shows the alpha toxin of Clostridium perfringens to
RT   contain both sphingomyelinase and lecithinase activities";
RL   Mol. Gen. Genet. 219:453-460(1989).
RN   [12]
RC   pCW3 from C.perfringens
RA   Rood J.I., Scott V.N., Duncan C.L.;
RT   "Identification of a transferable tetracycline resistance plasmid
RT   (pCW3) from Clostridium perfringens";
RL   Plasmid 1:563-570(1978).
RN   [13]
RC   pJIR39 from pCW3 & pUC18
RA   Abraham L.J., Rood J.I.;
RT   "Cloning and analysis of the Clostridium perfringens tetracycline
RT   resistance plasmid, pCW3";
RL   Plasmid 13:155-162(1985).
RN   [14]
RC   pPM20 from pUC13 & C.difficile tet gene
RA   Mullany P.;
RT   ;
RL   Unpublished (1988).
RN   [15]
RC   pAB124 from Bacillus tet gene
RC   pUB1730 from pAB124, Bacillus tet gene
RA   Eccles S.J., Chopra I.;
RT   "Biochemical and genetic characterization of the tet determinant
RT   of Bacillus plasmid pAB124";
RL   J. Bacteriol. 158:134-140(1984).
RN   [16]
RC   pSL1456 from E.coli tet gene
RC   pSL1501 from pSL1456, tet gene
RA   Marshall B., Morrissey S., Flynn P., Levy S.B.;
RT   "A new tetracycline-resistance determinant, class E, isolated from
RT   Enterobacteriaceae";
RL   Gene 50:111-117(1986).
RN   [17]
RC   RA1 from E.coli tet gene
RC   pWH801 from RA1, E.coli tet gene
RA   Unger R., Klock G., Hillen W.;
RT   "Nucleotide sequence of the repressor gene of the RA1 tetracycline
RT   resistance determinant: structural and functional comparison with
RT   three related Tet repressor genes";
RL   Nucleic Acids Res. 12:7693-7703(1984).
RN   [18]
RC   pCP1 from C.perfringens
RA   Smart J.L., Roberts T.A., Stringer M.F., Shah N.;
RT   "The incidence and serotypes of Clostridium perfringens on beef,
RT   pork and lamb carcasses";
RL   J. Appl. Bacteriol. 46:377-383(1979).
RN   [19]
RC   pSL100 from pKK232-8
RA   Li S.C., Squires C.L., Squires C.;
RT   "Antitermination of E. coli rRNA transcription is caused by a control
RT   region segment containing lambda nut-like sequences";
RL   Cell 38:851-860(1984).
CC   Deposited by: Julian I. Rood
CC   Shuttle expression vector permitting visual detection of recombinants
CC   in E. coli using lacZalpha complementation. Does not contain an intact
CC   beta-lactamase (bla) gene. [1]
CC   The genes for chloramphenicol and erythromycin resistance (catP and
CC   ermBP, respectively) were cloned from C. perfringens and can be used
CC   for selection in both C. perfringens and E. coli. [1]
CC   EcoRV and ScaI restriction sites can be used for cloning and
CC   insertional inactivation of catP and ermBP, respectively. [1]
CC   The standard reverse sequencing primer is not useful in this vector
CC   due to the duplication of the 181 bp region between pUC18 sites PvuII
CC   and EcoRI. [1]
CC   The vector contains the following restriction sites (position in bp):
CC   EcoRI--1; HindIII--57; PvuI--175; FspI--195; NarI--216; Tth111I--397;
CC   SpeI--3235; NheI--3549; NaeI--3653; HpaI--4206; EcoRV--4723;
CC   SmaI--4984; AvaII--5059; ScaI--5850. [1]
CC   The order of the major features in this plasmid is:  5' lacZ' -
CC   EcoRI/MCS/HindIII - 3' lacZ' - pIP404 ori - pIP404 rep - HpaI - catP
CC   - SmaI - AvaII - ermBP - pMB1 ori. [1]
CC   Location of the major features in this plasmid: lacZ' (nt 7345-310),
CC   MCS (nt 1-57), pIP404 ori (approx. nt 1190-1419), pIP404 rep gene (nt
CC   2702-1482), catP (nt 4830-4207), ermBP (nt 5902-5165), pMB1 ori
CC   (approx. nt 6780-7000). [1]
CC   Restriction digests of the clone give the following sizes (kb):
CC   EcoRI--7.4; HindIII--7.4; SmaI--5.0, 2.4. (ATCC staff)
CC   Medium is 1065 plus chloramphenicol (30 ug/ml).
CC   NCBI gi: 208130 & 144781
CC   NM (pJIR418)
CC   CM (yes)
CC   NA (ds-DNA)
CC   TP (circular)
CC   ST ()
CC   TY (plasmid)
CC   SP ()
CC   HO (E.coli)(E.coli DH5alpha)(Clostridium perfringens)
CC   CP ()
CC   FN (cloning)
CC   SE ()
CC   PA (pIP404)(pUC18)
CC   BR ()
CC   OF ()
CC   OR ()
FH   Key             Location/Qualifiers
FT   misc_feature    0..0
FT                   /note="1. C.perfringens, tet gene
FT                   -> pCW3 47000bp
FT                   1. pCW3 4000bp 45700..47000..2700, tet gene
FT                   2. pUC18 2686bp
FT                   -> pJIR39 6700bp
FT                   1. C.perfringens
FT                   -> pIP401 10200bp
FT                   1. pUC18 XbaI 2686bp 424..424
FT                   2. pIP401 XbaI-XbaI 8700bp, cat gene
FT                   -> pJIR45 11400bp
FT                   1. pUC18 remove HindIII-PstI 16bp 400..416,MCS/2670bp
FT                   2. pJIR45 HindIII-PstI 4300bp, cat gene
FT                   -> pJIR52 7000bp
FT                   1. pJIR52 HindIII 7000bp
FT                   BAL31 nuclease, remove 2800bp
FT                   -> pJIR62 4200bp [cat gene]
FT                   1. pUC18 EcoRI-DraI 1115bp 451..1566, ori
FT                   2. pJIR62 PvuII-EcoRI 1600bp, C.perfringens cat gene
FT                   -> pJIR274 2700bp
FT                   1. pJIR39 PstI-PstI 2900bp 45500..1600, tet gene
FT                   2. pUC18 PstI 2686bp 416..416, MCS
FT                   -> pJIR71 5600bp
FT                   1. pJIR274 large EcoRI-KpnI 2700bp
FT                   2. pJIR71 EcoRI-KpnI 800bp, C.perfringens tet gene
FT                   -> pJIR275 3500bp
FT                   1. pUC18 2686bp
FT                   2. C.perfringens 1200bp, ermP gene
FT                   -> pJIR122 3900bp
FT                   1. pJIR122 3900bp
FT                   -> pJIR208 3900bp
FT                   1. pJIR208 3900bp
FT                   -> pJIR229 3900bp
FT                   1. pJIR229 EcoRI-PstI 1200bp, ery gene
FT                   2. pJIR275 remove EcoRI-PstI 800bp, tet gene/2700bp
FT                   -> pJIR292 3900bp
FT                   1. pJIR292 HindIII 3900bp
FT                   2. pIP404 HindIII-HindIII 3100bp, ori
FT                   -> pJIR297 7100bp
FT                   1. pJIR297 Asp718I 7100bp
FT                   Klenow
FT                   -> pJIR335 7100bp
FT                   1. pJIR335 KpnI
FT                   Klenow
FT                   EcoRI
FT                   Klenow
FT                   -> pJIR403 7100bp [no KpnI, no EcoRI]
FT                   1. pJIR403 BamHI-PstI 7100bp
FT                   T4 DNA polymerase:T4 DNA polymerase
FT                   -> pJIR408 7100bp [no BamHI, no PstI]
FT                   1. pJIR408 HindIII 7100bp, between ori and cat
FT                   Klenow
FT                   -> pJIR415 7100bp
FT                   1. pJIR415 PvuII-EcoRV 5400bp, ori/cat/ery
FT                   2. pUC18 DraI-DraI 1975bp 2277..2686..1566,
FT                   \ MCS/lacZalpha
FT                   -> pJIR418 7358bp"
FT   misc_feature    1..57
FT                   /note="MCS EcoRI-SacI-KpnI-SmaI-BamHI-XbaI-SalI-
FT                   PstI-SphI-HindIII; from pUC18"
FT   misc_binding    1..57
FT                   /note="SIT unique EcoRI-SacI-KpnI-BamHI-XbaI-SalI-
FT                   PstI-SphI-HindIII"
FT   misc_binding    0..0
FT                   /note="SIT unique EcoRV"
FT   misc_binding    0..0
FT                   /note="SIT unique ScaI"
FT   rep_origin      1190..1419
FT                   /note="ORI C. perfringens pIP404 replication origin
FT                   [2], (#M32882)"
FT   CDS             complement(1482..2702)
FT                   /note="GEN C. perfringens replication enzyme gene
FT                   (rep), for pIP404 replication, partial, [2] (#M32882)"
FT   CDS             complement(4207..4830)
FT                   /note="ANT C. perfringens chloramphenicol
FT                   acetyltransferase gene (CATP), chloramphenicol
FT                   resistance gene (Cmr)"
FT   CDS             complement(5165..5902)
FT                   /note="GEN C. perfringens RNA methylase gene (ermBP),
FT                   for methylation of 23S rRNA [3] [4] (#M74769,#X58285)"
FT   rep_origin      6780..7000
FT                   /note="ORI E. coli pMB1 (ColE1 and pBR322);
FT                   E. coli pUC18 replication region oriEC"
FT   promoter        0..0
FT                   /note="PRO E. coli lac gene"
FT   CDS             join(7345..7358,1..310)
FT                   /note="GEN E. coli beta-galactosidase gene (lacZ')
FT                   alpha peptide, from pUC18"
SQ   Sequence 7358 BP; 2236 A; 1404 C; 1156 G; 2562 T; 0 other;
     gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagcttggc
     actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     acgttttcca atgatgagca cttttaaatt aaaaatgaag ttttaaaact tcatttttaa
     tttaaattaa aaatgaagtt ttatcaaaaa aatttccaat aatcccactc taagccacaa
     acacgcccta taaaatcccg ctttaatccc actttgagac acatgtaata ttactttacg
     ccctagtata gtgataattt tttacattca atgccacgca aaaaaataaa ggggcactat
     aataaaagtt ccttcggaac taactaaagt aaaaaattat ctttacaacc tccccaaaaa
     aaagaacagg tacaaagtac cctataatac aagcgtaaaa aaatgagggt aaaaataaaa
     aaataaaaaa ataaaaaaat aaaaaaataa aaaaaataaa aaaataaaaa aataaaaaaa
     taaaaaaata aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata
     aaaataaaaa aatataaaaa taaaaaaata taaaaataaa aaaataaaaa aatataaaaa
     taaaaaaata aaaaaatata aaaatatttt ttatttaaag tttgaaaaaa atttttttat
     attatataat ctttgaagaa aagaatataa aaaatgagcc tttataaaag cccatttttt
     ttcatatacg taatatgacg ttctaatgtt tttattggta cttctaacat tagagtaatt
     tctttatttt taaagccttt ttctttaagg gcttttattt tttttcttaa tacatttaat
     tcctcttttt ttgttgcttt tcctttagct tttaattgct cttgataatt ttttttacct
     ctaatatttt ctcttctctt atattccttt ttagaaatta ttattgtcat atatttttgt
     tcttcttctg taatttctaa taactctata agagtttcat tcttatactt atattgctta
     tttttatcta aataacatct ttcagcactt ctagttgctc ttataacttc tctttcactt
     aaatgttgtc taaacatact attaagttct aaaacatcat ttaatgcctt ctcaatgtct
     tctgtaaagc tacaaagata atatctatat aaaaataata taagctctct gtgtcctttt
     aaatcatatt ctcttagttc acaaagtttt attatgtctt gtattcttcc ataatataaa
     cttctttctc tataaatata atttattttg cttggtctac cctttttcct ttcatatggt
     tttaattcag gtaaaaatcc attttgtatt tctcttaagt cataaatata ttcgtactca
     tctaatatat tgactactgt ttttgattta gagtttatac ttcctggaac tcttaatatt
     ctggttgcat ctaaggcttg tctatctgct ccaaagtatt ttaattgatt atataaatat
     tcttgaaccg ctttccataa tggtaatgct ttactaggta ctgcatttat tatccatatt
     aaatacattc ctcttccact atctattaca tagtttggta taggaatact ttgattaaaa
     taattctttt ctaagtccat taatacctgg tctttagttt tgccagtttt ataataatcc
     aagtctataa acagtgtatt taactctttt atattttcta atcgcctaca cggcttataa
     aaggtattta gagttatata gatattttca tcactcatat ctaaatcttt taattcagcg
     tatttatagt gccattggct atatcctttt ttatctataa cgctcctggt tatccaccct
     ttacttctac tatgaatatt atctatatag ttctttttat tcagctttaa tgcgtttctc
     acttattcac ctccccttct gtaaaactaa gaaaattata tcatattttc aataattatt
     aactattctt aaactcttaa taaaaaatag agtaagtccc caattgaaac ttaatctatt
     ttttatgttt taatttatta tttttattaa aatattttaa actaaattaa atgattcttt
     ttaatttttt actatttcat tccataatat attactataa ttatttacaa ataatatttc
     ttcatttgta atatttagat gatttactaa ttttagtttt tatatattaa ataattaatg
     tataatttat ataaaaaatc aaaggagctt ataaattatg attatttcca aagatactaa
     agatttaatt ttttcaattt taacaatact ttttgtaata ttatgtttaa atttaattgt
     atttttttca tataataaag ccgttgaagt aaaccaatcc attttcctta tgatgttatt
     attaaattta agttttataa taatatcttt attatattta ttgtttttaa aaaaactagt
     gaaatttccg gctttattaa acttattttt aggaatttta ttttcatttt catctttaca
     ggatttgatt atatctttaa atatgtttta tcaaatatta tctttttcta aatttatata
     tatttttatt atatttatta ttatatatat tttattttta agtttctttc taacagctat
     taaaaagaaa cttaaaaata aaaacacgta ctctaaacca ataaataaaa ctatttttat
     tattgctgcc ttgattggaa tagtttttag taaaattaat ttcaatattc cacaatatta
     tattataagc tagctttgca ttgtactttt caatcgcttc acgaatgcgg ttatctccga
     aagataaagt cttttcatct tccttgatga agataagatt ttctccgtct ccgccggcag
     aattgaagcg gggtactacg gtatcgtctg cgtcatcttc cgttgtctga tagatgatag
     tcataggctc attttcttcc gtttcggtaa aggggatagg ttcgcccttt gagagcaggg
     cggcgatgga aagcattaac ttgcttttcc catcgcccgg atctccctgc aatagcgtaa
     ctttgccaaa cggaatatac ggataccaca gccactttac ttctttcggc tcgatttcac
     ttgccttgat gatttcaaga ggtacgctga aattcatttc gttttcattt agtttcattt
     tttcttgttc tccttttctc tgaaaatata aaaaccacag attgatacta aaaccttggt
     tgtgttgctt ttcggggctt aaatcaagga aaaatccttg ttttaagcct ttcaaaaaga
     aacacaaggt ctttgtacta acctgtggtt atgtataaaa ttgtagattt tagggtaaca
     aaaaacaccg tatttctacg atgtttttgc ttaaatactt gtttttagtt acagacaaac
     ctgaagttaa ctatttatca attcctgcaa ttcgtttaca aaacggcaaa tgtgaaatcc
     gtcacatact gcgtgatgaa cttgaattgc caaaggaagt ataattttgt tatcttcttt
     ataatatttc cccatagtaa aaataggaat caaataatca tatcctttct gcaaattcag
     attaaagcca tcgaaggttg accacggtat catagataca ttaaaaatgt tttccggagc
     atttggcttt ccttccattc tatgattgtt tccataccgt tgcgtatcac tttcataatc
     tgctaaaaat gatttaaagt cagacttaca ctcagtccaa aggctggaaa atgtttcagt
     atcattgtga aatattgtat agcttggtat catctcatca tatatcccca attcaccatc
     ttgattgatt gccgtcctaa actctgaatg gcggtttaca atcattgcaa tataataaag
     cattgcagga tatagtttca ttcccttttc ctttatttgt gtgatatcca ctttaacggt
     catgctgtat gtacaaggta cacttgcaaa gtagtggtca aaatactctt ttctgttcca
     actattttta tcaatttttt caaataccat ctaagttccc tctcaaattc aagtttatcg
     ctctaatgaa caaagatatt ataccacatt tttgtgaatt tttcaacttg cccacttcga
     ctgcactccc gacttaataa cttcttgaac acttgccgaa aaagaaaaac tgccgggtac
     gtacccggga tcgatccccg ccgagcgctt agtgggaatt tgtacccctt atcgatacaa
     attccccgta ggcgctaggg acctctttag ctccttggaa gctgtcagta gtatacctaa
     taatttatct acattccctt tagtaacgtg taactttcca aatttacaaa agcgactcat
     agaattattt cctcccgtta aataatagat aactattaaa aatagacaat acttgctcat
     aagtaacggt acttaaattg tttactttgg cgtgtttcat tgcttgatga aactgatttt
     tagtaaacag ttgacgatat tctcgattga cccattttga aacaaagtac gtatatagct
     tccaatattt atctggaaca tctgtggtat ggcgggtaag ttttattaag acactgttta
     cttttggttt aggatgaaag cattccgctg gcagcttaag caattgctga atcgagactt
     gagtgtgcaa gagcaaccct agtgttcggt gaatatccaa ggtacgcttg tagaatcctt
     cttcaacaat cagatagatg tcagacgcat ggctttcaaa aaccactttt ttaataattt
     gtgtgcttaa atggtaagga atactcccaa caattttata cctctgtttg ttagggaatt
     gaaactgtag aatatcttgg tgaattaaag tgacacgagt attcagtttt aatttttctg
     acgataagtt gaatagatga ctgtctaatt caatagacgt tacctgttta cttattttag
     ccagtttcgt cgttaaatgc cctttacctg ttccaatttc gtaaacggta tcggtttctt
     ttaaattcaa ttgttttatt atttggttga gtactttttc actcgttaaa aagttttgag
     aatattttat atttttgttc atgtaatcac tccttcttaa ttacaaattt ttagcatcta
     atttaacttc aattcctatt atacaaaatt ttaagatact gcactatcaa cacactctta
     agtttgcttc taagtcttat ttccataact tcttttacgt ttccgggtac aattcgtaat
     catgtcatag ctgtttcctg tgtgaaattc ttatccgctc acaattccac acaacatacg
     agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccaga aaacttcatt
     tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     aacaatttca cacaggaaac agctatgacc atgattac