pAf-CRISPR-phoA

LOCUS       pAf-CRISPR-phoA       13733 bp DNA     circular SYN 25-AUG-2024
DEFINITION  CRISPR vector used with pAf-CRISPR-ctfR1 (#207992) to delete both 
            aflatoxin and cyclopiazonic acid gene clusters of Aspergillus 
            flavus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13733)
  AUTHORS   Chang PK
  TITLE     Creating large chromosomal segment deletions in Aspergillus flavus 
            by a dual CRISPR/Cas9 system: Deletion of gene clusters for 
            production of aflatoxin, cyclopiazonic acid, and ustiloxin B.
  JOURNAL   Fungal Genet Biol. 2023 Dec 27;170:103863. doi: 
            10.1016/j.fgb.2023.103863.
  PUBMED    38154756
REFERENCE   2  (bases 1 to 13733)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2024 from SnapGene Server 7.0.3
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Fungal 
            Genet Biol."; date: "2023-12-27"; volume: "170:103863. doi"; pages: 
            " 10.1016/j.fgb.2023.103863"
FEATURES             Location/Qualifiers
     source          1..13733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        3129..3204
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(3612..3632)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3645..7748)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(3645..7748)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(7779..7799)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          7820..7935
                     /gene="gpdA"
                     /label=gpdA intron
                     /note="intron from the Aspergillus nidulans 
                     glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al.,
                     1990)"
     primer_bind     complement(9056..9072)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9056..9072)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9069..9091)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9080..9096
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(9104..9134)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9149..9170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9287..9304)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9458..10046)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9538..9557)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(10217..11077)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     10840..10859
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     gene            11095..13102
                     /product="pyrithiamine resistance gene of Aspergillus 
                     oryzae (Kubodera et al., 2000)"
                     /label=ptrA
                     /note="useful as a dominant selectable marker in 
                     Aspergillus"
     CDS             complement(11408..11419)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(11817..12153,12212..12858)
                     /codon_start=1
                     /gene="ptrA of Aspergillus oryzae"
                     /product="pyrithiamine resistance protein (Kubodera et al.,
                     2000)"
                     /label=ptrA
                     /note="useful as a dominant selectable marker in 
                     Aspergillus"
                     /translation="MSPPAAIYEPTVAATGLKGKVVVSETVPVEGASQTKLLDHFGGKW
                     DEFKFAPIRESQVSRAMTRRYFEDLDKYAESDVVIVGAGSCGLSTAYVLAKARPDLKIA
                     IVEASVSPGGGAWLGGQLFSAMVMRRPAEVFLNELGVPYEEDANPNYVVVKHASLFTST
                     LMSKVLSFPNVKLFNATAVEDLITRPTENGNPQIAGVVVNWTLVTLHHDDHSCMDPNTI
                     NAPVIISTTGHDGPFGAFCAKRLVSMGSVDKLGGMRGLDMSSAEDAIVKNTREVTKGLI
                     IGGMELSEIDGFNRMGPTFGAMVLSGVKAAEEALKVFDERQRECAE"
     primer_bind     complement(13221..13242)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aaaaaaaaaa aacaagacag ccacatatta gggagactag ttagaagcta gttccaggac
       61 taggaaaata aaagacaatg ataccacagt ctagttgaca actagataga ttctagattg
      121 aggccaaagt ctctgagatc caggttagtt gcaactaata ctagttagta tctagtctcc
      181 tataactctg aagctagaat aacttactac tattatcctc accactgttc agctgcgcaa
      241 acggagtgat tgcaaggtgt tcagagacta gttattgact agtcagtgac tagcaataac
      301 taacaaggta ttaacctacc atgtctgcca tcaccctgca cttcctcggg ctcagcagcc
      361 ttttcctcct cattttcatg ctcattttcc ttgtttaaga ctgtgactag tcaaagacta
      421 gtccagaacc acaaaggaga aatgtcttac cactttcttc attgcttgtc tcttttgcat
      481 tatccatgtc tgcaactagt tagagtctag ttagtgacta gtccgacgag gacttgcttg
      541 tctccggatt gttggaggaa ctctccaggg cctcaagatc cacaacagag ccttctagaa
      601 gactggtcaa taactagttg gtctttgtct gagtctgact tacgaggttg catactcgct
      661 ccctttgcct cgtcaatcga tgagaaaaag cgccaaaact cgcaatatgg ctttgaacca
      721 cacggtgctg agactagtta gaatctagtc ccaaactagc ttggatagct tacctttgcc
      781 ctttgcgttg cgacaggtct tgcagggtat ggttcctttc tcaccagctg atttagctgc
      841 cttgctaccc tcacggcgga tctgcataaa gagtggctag aggttataaa ttagcactga
      901 tcctaggtac ggggctgaat gtaacttgcc tttcctttct catcgcgcgg caagacaggc
      961 ttgctcaaat tcctaccagt cacaggggta tgcacggcgt acggaccact tgaactagtc
     1021 acagattagt tagcaactag tctgcattga atggctgtac ttacgggccc tcgccattgt
     1081 cctgatcatt tccagcttca ccctcgttgc tgcaaagtag ttagtgacta gtcaaggact
     1141 agttgaaatg ggagaagaaa ctcacgaatt ctcgacaccc ttagtattgt ggtccttgga
     1201 cttggtgctg ctatatatta gctaatacac tagttagact cacagaaact tacgcagctc
     1261 gcttgcgctt cttggtagga gtcggggttg ggagaacagt gccttcaaac aagccttcat
     1321 accatgctac ttgactagtc agggactagt caccaagtaa tctagatagg acttgccttt
     1381 ggcctccatc agttccttca tagtgggagg tccattgtgc aatgtaaact ccatgccgtg
     1441 ggagttcttg tccttcaagt gcttgaccaa tatgtttctg ttggcagagg gaacctgtca
     1501 actagttaat aactagtcag aaactagtat agcagtagac tcactgtacg cttgaggcat
     1561 cccttcactc ggcagtagac ttcatatgga tggatatcag gcacgccatt gtcgtcctgt
     1621 ggactagtca gtaactaggc ttaaagctag tcgggtcggc ttactatctt gaaatccggc
     1681 agcgtaagct ccccgtcctt aactgcctcg agatagtgac agtactctgg ggactttcgg
     1741 agatcgttat cgcgaatgct cggcatacta atcgttgact agtcttggac tagtcccgag
     1801 caaaaaggat tggaggagga ggaggaaggt gagagtgaga caaagagcga aataagagct
     1861 tcaaaggcta tctctaagca gtatgaaggt taagtatcta gttcttgact agatttaaaa
     1921 gagatttcga ctagttatgt acctggagtt tggatatagg aatgtgttgt ggtaacgaaa
     1981 tgtaaggggg aggaaagaaa aagtcggtca agaggtaact ctaagtcggc cattcctttt
     2041 tgggaggcgc taaccataaa cggcatggtc gacttagagt tagctcaggg aatttaggga
     2101 gttatctgcg accaccgagg aacggcggaa tgccaaagaa tcccgatgga gctctagctg
     2161 gcggttgaca accccacctt ttggcgtttc tgcggcgttg caggcgggac tggatacttc
     2221 gtagaaccag aaaggcaagg cagaacgcgc tcagcaagag tgttggaagt gatagcatga
     2281 tgtgccttgt taactaggtc aaaatctgca atatgcttga tgttatccaa agtgtgagag
     2341 aggaaggtcc aaacatacac gattgggaga gggcctaggt ataagagttt ttgagtagaa
     2401 cgcatgtgag cccagccatc tcgaggagat taaacacggg ccggcatttg atggctatgt
     2461 tagtacccca atggaaagcc tgagagtcca gtggtcgcag ataactccct aaattccctg
     2521 agctaactct aagtcgacca tgccgtttat ggttagcgcc tcccaaaaag gaatggccga
     2581 cttagagtta cctcttgacc gactgcagtt ctctttagaa ttcaactgtg ggttttgctt
     2641 tttgcttcat tctctttgtc ttctccatct ttgatcaaat cctggacttt ctcaatcccc
     2701 agctaattca atcatagtca gttttctatt tttattattt ctttttcttt tgaaatgtga
     2761 ttaacaacca gtctgttata tatcttgtac ccagattacg cccaactcgt gctcctcagc
     2821 cacaaagata ctcaattgat agccaagata catacatacc acaaagtaag gactccatgc
     2881 attgagtatt actcatcgta ttctagacta ctccaaaact cagcacatag acaaacaata
     2941 cgaacctcgt ctaagggtga ttcagaggcg gcaaagcggg gttttcgcat ttgatgttcc
     3001 tggcacttat gtaagcccac gcttcccgct caactaaacc atcagccaat cagactgctc
     3061 agatttatct tttgaagggt atataaatca ttgtaaagaa gaacaagtgt catccttaga
     3121 cgacactcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     3181 gaaaaagtgg caccgagtcg gtgctttttt tttgagcatt tatcagcttg atatagaggt
     3241 aggaatgtat ggaggtgcag aatggctatt ttgttattgg agcgggttcg aaacggaggg
     3301 caggagactt tttttaaata tgtggtaccg ttactaggtt gcagtcaatg cattaatgca
     3361 ttgcagatga gctgtatctg gaagaggtaa acccgaaacg cgttttattc ttgttgacat
     3421 ggagctatta aatcactaga aggcactctt tgctgcttgg acaaatgaac gtatcttatc
     3481 gagatcctga acaccatttg tctcaactcc ggagctgaca tcgacaccaa cgatcttata
     3541 tccagattcg tcaagctgtt tgatgatttc agtaacgtta agtggatccg gtccggaatt
     3601 cccctagctc agaccttacg cttcttcttg gggtcagcac gggagtcacc accgagctga
     3661 gagaggtcga tacgagtctc gtagagaccg gtaatggact ggtggatgag agtggcgtcg
     3721 aggacctcct tggtagaggt gtaacgctta cggtcgatgg tggtgtcgaa gtacttgaag
     3781 gcagcagggg caccaaggtt ggtgagagtg aagaggtgga tgatgttctc agcctgctca
     3841 cggatgggct tgtcacggtg cttgttgtaa gcggagagga ccttatcgag gttagcgtcg
     3901 gcgaggatga cacgcttgga gaactcggag atctgctcga tgatctcatc aaggtagtgc
     3961 ttgtgctgct cgacgaagag ctgcttctgc tcgttgtctt cgggagaacc cttgagcttc
     4021 tcgtagtggg aggcgaggta gaggaagttg acgtacttgg aggggagagc gagctcgtta
     4081 cccttctgga gctcaccggc ggaagcgagc atacgcttac gaccgttctc aagctcgaag
     4141 agggagtact tgggcagctt gatgatgagg tccttcttga cctccttgta acccttggcc
     4201 tcgaggaagt cgatggggtt cttctcgaag gaggaacgct ccatgatggt gatgccgagg
     4261 agctccttga cggacttgag cttcttagac ttacccttct cgaccttggc gacaacgagg
     4321 acggagtaag cgacagtggg agagtcgaaa ccgccgtact tcttggggtc ccagtccttc
     4381 ttgcgagcga tgagcttgtc ggagttacgc ttggggagga tagattcctt ggagaagcca
     4441 ccggtctgga cctcggtctt cttgacgatg ttgacctggg gcatggagag gaccttacgg
     4501 acagtggcga agtcacgacc cttgtcccag acgatctcac cagtctcacc gttggtctcg
     4561 atgaggggac gcttacggat ctcaccgttg gcgagggtga tctcggtctt gaagaagttc
     4621 atgatgttgg agtagaagaa gtacttagcg gtggccttac cgatctcctg ctcggactta
     4681 gcgatcatct tacgaacatc gtagaccttg tagtcaccgt agacgaactc ggactccagc
     4741 ttggggtact tcttgataag agcagtgcca acgacagcgt tgaggtaggc atcgtgggcg
     4801 tggtggtagt tgttgatctc acggaccttg tagaactgga agtccttacg gaagtcggag
     4861 acgagcttag acttgagagt gataaccttg acctcacgga tgagcttgtc gttctcatcg
     4921 tacttggtgt tcatgcgaga gtcgagaatc tgggcgacgt gcttggtgat ctgacgagtc
     4981 tcaacaagct gacgcttgat gaagccggcc ttgtcgagct cagagaggcc accacgttca
     5041 gccttagtga ggttatcgaa cttacgctgg gtgatgagct tggcgttgag aagctgacgc
     5101 cagtagttct tcatcttctt gacaacctct tcggagggga cgttgtcaga cttgccacgg
     5161 ttcttatcgg aacgggtgag gaccttgttg tcgatagagt catccttgag gaaggactgg
     5221 gggacgatgt gatcgacgtc gtagtcggag agacggttga tgtcgagctc ctgatcaacg
     5281 tacatgtcac ggccgttctg gaggtagtag aggtagagct tctcgttctg aagctgggtg
     5341 ttctcgacgg ggtgctcctt aaggatctgg gagccgagtt ccttgatacc ctcctcgata
     5401 cgcttcatac gttcacgaga gttcttctga cccttctggg tagtctggtt ctcacgggcc
     5461 atctcgataa cgatgttctc gggcttgtga cgacccataa ccttgacgag ctcatcgacg
     5521 accttaacgg tctggaggat acccttcttg atagcggggg aaccggcgag gttagcgatg
     5581 tgctcgtgaa gggagtcacc ctgaccggaa acctgagcct tctggatgtc ctccttgaag
     5641 gtgagagagt cgtcgtggat gagctgcatg aagttacggt tggcgaaacc atcggactta
     5701 aggaagtcga ggatagtctt accagactgc ttgtcacgga tgccgttgat cagcttacgg
     5761 gagagacggc cccaaccagt gtaacgacga cgcttgagct gcttcatgac cttgtcgtcg
     5821 aagaggtgag cgtaagtctt gagacgttct tcgatcatct cacgatcctc gaagagggta
     5881 agggtgagga cgatgtcctc aaggatgtcc tcgttctcct cgttgtcgag gaagtccttg
     5941 tccttgatga tcttgaggag atcgtggtag gtgccgaggg aagcgttgaa acgatcctcg
     6001 acaccggaga tctcaacgga gtcgaagcac tcgatcttct tgaagtagtc ttccttgagc
     6061 tgcttgacgg taaccttacg gttggtcttg aagaggaggt cgacgatagc cttcttctgc
     6121 tcaccagaga ggaaggcggg cttacgcata ccttcggtga cgtacttgac cttggtgagc
     6181 tcgttgtaaa cagtgaagta ctcgtaaagg agagagtgct taggaaggac cttctcgtta
     6241 gggaggttct tgtcgaagtt agtcatacgc tcgatgaagg actgggcaga ggcaccctta
     6301 tcgacgactt cctcgaagtt ccagggagtg atggtctctt cggacttacg agtcatccag
     6361 gcgaaacggg agttaccacg ggcgaggggg ccaacgtagt aggggatacg gaaggtgagg
     6421 atcttctcga tcttctcacg gttgtccttg aggaaggggt agaagtcctc ctgacgacgg
     6481 aggatagcgt ggagttcgcc aaggtggatc tggtggggga tggaaccgtt gtcgaaagta
     6541 cgctgcttac ggaggagatc ctcacggttg agcttgacga ggagctcctc ggtgccgtcc
     6601 atcttctcga ggatgggctt gatgaacttg tagaactcct cctgggaagc accgccgtcg
     6661 atgtaaccgg cgtaaccgtt cttggactga tcgaagaaga tctccttgta cttctcaggg
     6721 agctgctgac ggacaagggc cttgagaaga gtgaggtcct ggtggtgctc atcgtaacgc
     6781 ttgatcatag aagcggagag gggagccttg gtgatctcgg tgttgacacg gaggatatcg
     6841 gagaggagga tggcgtcgga gaggttctta gcggcaagga agaggtcagc gtactggtca
     6901 ccgatctggg cgaggaggtt gtcgagatcg tcatcgtagg tgtccttgga gagctggagt
     6961 ttggcatcct cggcgaggtc gaagttggac ttgaagttgg gggtgagacc gagagagagg
     7021 gcgataaggt taccgaagag accgttcttc ttctcaccag ggagctgagc gatgaggttc
     7081 tcgagacgac gggacttgga gagacgagcg gagaggatgg ccttggcgtc aacaccggaa
     7141 gcgttgatgg ggttctcttc gaagagctgg ttgtaagtct gaacgagctg gatgaagagc
     7201 ttgtcgacat cggagttgtc agggttgagg tcaccctcga tgaggaagtg accacggaac
     7261 ttgatcatgt gggcgagggc gaggtagatg agacgaaggt cagccttatc agtagagtcg
     7321 accagcttct tacggaggtg gtagatggtg gggtacttct cgtggtaggc gacctcgtcg
     7381 acgatgttac cgaagatggg gtgacgctcg tgcttcttat cctcctcgac gaggaaggac
     7441 tcctcgagac ggtggaagaa ggagtcatcg accttagcca tctcgttgga gaagatctcc
     7501 tggaggtagc agatacggtt cttacgacgg gtgtaacgac gacgggcggt acgcttgaga
     7561 cgggtggcct cggcggtctc accggagtcg aagaggaggg caccgatgag gttcttcttg
     7621 atggagtgac ggtcggtgtt accgaggacc ttgaacttct tggaggggac cttgtactcg
     7681 tcggtgatga cggcccaacc gacggagttg gtgccgatgt cgagaccgat ggagtacttc
     7741 ttgtccatgc ggccggctgc tgggactccg tggataccga ccttccgctt cttctttggg
     7801 gccatgatca tgcggccgcc tgctcaagcg gggtagctgt tagtcaagct gcgatgaagt
     7861 gggaaagctc gaactgaaag gttcaaagga ataagggatg ggaaggatgg agtatggatg
     7921 tagcaaagta cttacttagg ggaaataaag gttcttggat gggaagatga atatactgaa
     7981 gatgggaaaa gaaagagaaa agaaaagagc agctggtggg gagagcagga aaatatggca
     8041 acaaatgttg gactgacgca acgaccttgt caaccccgcc gacacaccgg gcggacagac
     8101 ggggcaaagc tgcctaccag ggactgaggg acctcagcag gtcgagtgca gagcaccgga
     8161 tgggtcgact gccagcttgt gttcccggtc tgcgccgctg gccagctcct gagcggcctt
     8221 tccggtttca tacaccgggc aaagcaggag aggcacgata tttggacgcc ctacagatgc
     8281 cggatgggcc aattagggag cttacgcgcc gggtactcgc tctacctact tcggagaagg
     8341 tactatctcg tgaatctttt accagatcgg aagcaattgg acttctgtac ctaggttaat
     8401 ggcatgctat ttcgccgacg gctatacacc cctggcttca cattctcctt cgcttactgc
     8461 cggtgattcg atgaagctcc atattctccg atgatgcaat agattcttgg tcaacgaggg
     8521 gcacaccagc ctttccactt cggggcggag gggcggccgg tcccggatta ataatcatcc
     8581 actgcacctc agagccgcca gagctgtctg gcgcagtggc gcttattact cagcccttct
     8641 ctctgcgtcc gtccgtctct ccgcatgcca gaaagagtca ccggtcactg tacagagctc
     8701 acgagttcgt cacatttttc tacaaatggt ggaggcggcg gattttaggc tcaagtcatg
     8761 accctctggg tcactccaga atcagctagg tcaacgaata aggatgattc tataggaaga
     8821 tccaggcacc ggtcaaccat gatctggaca gatttgggag ctcggtataa gctctccacc
     8881 atcttattct gtatagttta ggcttaaagt ttatccagga gatgttgctg aagtcgattt
     8941 gagtccactt cctcactggt agctatacga ctttgatggt cgttgtaggg gctgtattag
     9001 gtctcgatca aacacaaata gaattaaatg gtactcgaag cttggcgtaa tcatggtcat
     9061 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     9121 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     9181 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     9241 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     9301 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     9361 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     9421 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     9481 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     9541 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     9601 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     9661 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     9721 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     9781 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     9841 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     9901 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     9961 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    10021 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    10081 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    10141 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    10201 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    10261 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    10321 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    10381 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    10441 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    10501 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    10561 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    10621 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    10681 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    10741 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    10801 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    10861 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    10921 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    10981 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    11041 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatgggcaa
    11101 ttgattacgg gatcccattg gtaacgaaat gtaaaagcta ggagatcgtc cgccgatgtc
    11161 aggatgattt cacttgtttc ttgtccggct caccggtcaa agctaaagag gagcaaaagg
    11221 aacggataga atcgggtgcc gctgatctat acggtatagt gcccttatca cgttgactca
    11281 acccatgcta tttaactcaa cccctccttc tgaaccccac catcttcttc cttttcctct
    11341 catcccacac aattctctat ctcagatttg aattccaaaa gtcctcggac gaaactgaac
    11401 aagtcttcct cccttcgata aacctttggt gattggaata actgaccatc ttctatagtt
    11461 cccaaaccaa ccgacaatgt aaatacactc ctcgattagc cctctagagg gcatacgatg
    11521 gaagtcatgg aatacttttg gctggactct cacaatgatc aaggtatctt aggtaacgtc
    11581 tttggcgtgg gccggtgttc gttcccagtc atcgatgcat tcacatgccc tccctaagct
    11641 gggccctaga ctctaggatc ctagtctaga aggacatggc atcgatggac tgggttcgtt
    11701 ctgagattat acggctaaaa cttgatctgg ataataccag cgaaaagggt catgccttct
    11761 ctcgttcttc ctgttgatgg aatggctaac agatgatagt cattgcaact tgaaacatgt
    11821 ctcctccagc tgccatctac gaacccactg tggccgctac cggcctcaag ggtaaggtcg
    11881 tggtttctga gaccgtcccc gttgagggag cttctcagac caagctgttg gaccatttcg
    11941 gtggcaagtg ggacgagttc aagttcgccc ctatccgcga aagccaggtc tctcgtgcca
    12001 tgaccagacg ttactttgag gacctggaca agtacgctga aagtgacgtt gtcattgttg
    12061 gtgctggttc ctgcggtctg agcactgcgt acgtcttggc caaggctcgt ccggacctga
    12121 agattgctat cgtcgaggcc agcgtctctc ctggtcagta gtccatgatg gattgccttg
    12181 cactcagctt tccggaacta acgtgcaata ggtggcggtg cctggttggg tggccaactc
    12241 ttttctgcta tggtcatgcg ccgtcccgcg gaagtcttcc tgaacgagct gggtgttcct
    12301 tacgaagagg acgcaaaccc caactacgtt gtcgtcaagc acgcctccct gtttacctcg
    12361 acactcatgt cgaaggttct ctccttcccc aatgtcaagc tcttcaatgc taccgctgtt
    12421 gaggacttga tcacccgtcc gaccgagaac ggcaaccccc agattgctgg tgttgtcgtc
    12481 aactggacgc tggtcaccct tcaccacgat gatcactcct gcatggaccc caacactatc
    12541 aacgctcctg tcatcatcag taccactggt cacgatgggc cattcggcgc cttctgtgcg
    12601 aagcgcttgg tgtccatggg cagcgtcgac aagctaggtg gcatgcgtgg tctcgacatg
    12661 agctcggccg aggatgccat cgtcaagaac acccgcgagg ttactaaggg cttgataatc
    12721 ggcggtatgg agctgtctga aattgatggc tttaaccgca tgggccctac cttcggtgcc
    12781 atggttctca gtggtgtcaa ggctgccgag gaggcattga aggtgttcga cgagcgtcag
    12841 cgcgagtgtg ctgagtaaat gactcactac ccgaatgggt tcagtgcatg aaccggattt
    12901 gtcttacggt ctttgacgat aggggaatga tgattatgtg atagttctga gatttgaatg
    12961 aactcgttag ctcgtaatcc acatgcatat gtaaatggct gtgtcccgta tgtaacggtg
    13021 gggcattcta gaataattat gtgtaacaag aaagacagta taatacaaac aaagatgcaa
    13081 gagcggctca tcgtcacccc attttgttaa aattcgcgtt aaatttttgt taaatcagct
    13141 cattttttaa ccaatagacc gaaatcggca aaatccctta taaatcaaaa gaatagcccg
    13201 agatagagtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
    13261 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
    13321 ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
    13381 gcccccgatt tagagcttga cggggaaagc cagcttattt tttgtatact gttttgtgat
    13441 agcacgaagt ttttccacgg tatcttgtta aaaatatata tttgtggcgg gcttacctac
    13501 atcaaattaa taagagacta attataaact aaacacacaa gcaagctact ttagggtaaa
    13561 agtttataaa tgcttttgac gtataaacgt tgcttgtatt tattattaca attaaaggtg
    13621 gatagaaaac ctagagacta gttagaaact aatctcaggt ttgcgttaaa ctaaatcaga
    13681 gcccgagagg ttaacagaac ctagaagggg actagatatc cgggtaggga aac
//