LOCUS       sequence-353319-       12088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing optimized Cas9 and NAT marker and sgRNA targeting
            ADE2 locus in D. hansenii.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12088)
  AUTHORS   Strucko T, Andersen NL, Mahler MR, Martinez JL, Mortensen UH
  TITLE     A CRISPR/Cas9 method facilitates efficient oligo-mediated gene 
            editing in Debaryomyces hansenii.
  JOURNAL   Synth Biol (Oxf). 2021 Oct 12;6(1):ysab031. doi: 
            10.1093/synbio/ysab031. eCollection 2021.
  PUBMED    34746438
REFERENCE   2  (bases 1 to 12088)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Synth Biol 
            (Oxf)."; date: "2021-10-12"; volume: "6(1):ysab031. doi"; pages: " 
            10.1093/synbio/ysab031. eCollection 2021"
COMMENT     Sequence Label: pDIV495
FEATURES             Location/Qualifiers
     source          1..12088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      829..1026
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1089..1122
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1192..1225)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(1695..1712)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1695..1711)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1810..1827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1981..2569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2061..2080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2691..3482,3483..3551))
                     /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     3314..3333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3552..3643)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3685..3704
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3710..3729
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    3936..3969
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_RNA        4992..5067
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             6759..10862
                     /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             6759..10862
                     /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             10875..10895
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10899..10919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10923..10943
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10944..11009
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(11090..11108)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      11120..11308
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 ttcctggtga aaaaccgcca caaattgacc atgacagtgc ttgctcggag ttttcagcgg
       61 agggcgtgcg cccgggcaaa catagtgcat tgtgatgtag gcggcaaata tatgccggat
      121 acgcgcttat ggagcggatt gccccggcgg agaaaagtat ataagtagcc agcgcaaggc
      181 ccccatttct agagaatttt tcccttcaat tgattacaac ttcaattaac tacaaacaaa
      241 acaaacaacc atgggaacca ccttggatga cactgcttat cgataccgaa catctgtacc
      301 tggtgatgct gaagctatcg aagcccttga cgggtcattc acaactgaca ccgtctttag
      361 agttacagct actggggatg ggtttacatt gcgagaagta ccagtagatc ctcctttgac
      421 caaagtattc ccagatgacg aaagtgacga tgaatccgat gacggtgagg acggtgaccc
      481 agactcaaga acatttgtcg cttatggaga cgacggtgat ttggccggtt tcgtagttgt
      541 atcctattct ggatggaacc gaagacttac tgtcgaggat atagaggttg ccccagaaca
      601 tcgtgggcac ggtgttgggc gagcccttat gggattagct acagaatttg cccgtgaacg
      661 tggggctgga catttatggt tagaagtcac caatgttaat gcaccagcta tacatgcata
      721 tcgaagaatg gggttcacac tttgtggatt ggatacagct ttatacgatg gtacagcctc
      781 agacggagaa caagctttat acatgtctat gccttgtcca taatcagtac tgacaataaa
      841 aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta
      901 ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat
      961 gcgaagttaa gtgcgcagaa agtaatatca tgcgttaatc gtatgtgaat gctggtcgct
     1021 atactgctgt cgattcgata ctaacgccgc catccagtgt cgaaaacgag ctctcgagaa
     1081 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtgat atcagatcca
     1141 ctagtggcca ttaagacctc agcgcggccg caaatttaaa taaaatgaag tgaagttcct
     1201 atactttcta gagaatagga acttctatag tgagtcgaat aagggcgaca caaaatttat
     1261 tctaaatgca taataaatac tgataacatc ttatagtttg tattatattt tgtattatcg
     1321 ttgacatgta taattttgat atcaaaaact gattttccct ttattatttt cgagatttat
     1381 tttcttaatt ctctttaaca aactagaaat attgtatata caaaaaatca taaataatag
     1441 atgaatagtt taattatagg tgttcatcaa tcgaaaaagc aacgtatctt atttaaagtg
     1501 cgttgctttt ttctcattta taaggttaaa taattctcat atatcaagca aagtgacagg
     1561 cgcccttaaa tattctgaca aatgctcttt ccctaaactc cccccataaa aaaacccgcc
     1621 gaagcgggtt tttacgttat ttgcggatta acgattactc gttatcagaa ccgcccaggg
     1681 ggcccgagct taagactggc cgtcgtttta caacacagaa agagtttgta gaaacgcaaa
     1741 aaggccatcc gtcaggggcc ttctgcttag tttgatgcct ggcagttccc tactctcgcc
     1801 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1861 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1921 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1981 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2041 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2101 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2161 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2221 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2281 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2341 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtgggc
     2401 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2461 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2581 gatcttttct acggggtctg acgctcagtg gaacgacgcg cgcgtaactc acgttaaggg
     2641 attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt aatgctctgc ttaccaatgc
     2701 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2761 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagcgctgcg
     2821 atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa ccagccagcc
     2881 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2941 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3001 atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3061 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3121 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3181 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3241 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3301 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3361 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3421 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3481 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3541 tgaatactca tattcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3601 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cagtgttaca
     3661 accaattaac caattctgaa cattatcgcg agcccattta tacctgaata tggctcataa
     3721 caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     3781 agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga gagtagggaa
     3841 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgcccgggct
     3901 aattatgggg tgtcgccctt attcgactct atagtgaagt tcctattctc tagaaagtat
     3961 aggaacttct gaagtgggga tttaaatgcg gccgcgctga gggtttaatg ggcttagtta
     4021 gccttgcttg tgactttgca gctttatcca tgaacggatc atacgattgt atttctgaat
     4081 ttgtcatagt gactttaagt tgttctttat ttggtatgat tctatttttt aggactttct
     4141 aaaaaatcaa tttaaatttt ggaaaaccat ttgtggtttt tcttaaatga ttgcaaaatt
     4201 attaaaaaat cgttaaaaaa atattcagtg taaaaagact agcattcatg atctcaacat
     4261 ctttggataa tatcagaatg agaaagaaca gatacgcagt acgttttttg gtgagctctt
     4321 tgcacttctt tagttctttc catcaatatc agttttttaa acttttagga ctaaaagtga
     4381 tgtttaactt caaaatgttt aaaattttgt tcttcccgac gttcattaag aatactaata
     4441 cactttaata attattttaa tattttgttc taaataatga cttttaatta aaaaagataa
     4501 aatataaaaa catcataata actcaccaga ggttaagaac aaaaaaacaa attagatatc
     4561 tgctaatcca atatagttaa atcaatcttt ccttggtata atgggtatat tacatatatt
     4621 tcaaggaccg acactcctac caaatatcta aaatttacca tattaacata acatgtatat
     4681 aaacgtcaaa tcataatcag cactagcgga atgcgtgcga tctgaacctc cccaggagtc
     4741 ataaataata gtcagatctt tcaggctcat cttgttctct ccatgttctt gcatataaac
     4801 atatcagacc gaacatatct cacttttttt catttttcta tgctgtgatg gcgtatggca
     4861 gaagcgcgca gattgtgtac ttttgattct ttattacagt ttaagtgtcc ggttcgatct
     4921 ctgcggtcgc aaacttgtga atttgagtaa tcaattcttc aagtttcagc aagctaagca
     4981 gattaatgca tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     5041 cttgaaaaag tggcaccgag tcggtgcttt ttttctcgag tttttttatc gagtgtttaa
     5101 ggataatgat aactgaagag aagaattagt tttgccgcca ccgcgggttt gcctctgatt
     5161 aaataaaaaa aagctggtgc ttttttttct tttataggaa catcttgaat atatgaacta
     5221 attaaatgat aattttttac ccatctttac tcttaatcac tgagctgcag tcaaagaaaa
     5281 agggatacag cacctggtga agagatgaac ggagactaac ttagacgcgt tgattctttt
     5341 caattgcaca ttttattaat cgatgctaac gtctatttac atatattctt tagagatatt
     5401 atctagggct tcaaataatc tctggacagc aataaaagtc tcttcaaaag tattgtataa
     5461 cggcaatggg gctaatctga ttacatctgg tcttctttcg tcacagatta tagcatgatc
     5521 atgcaagtac gcattaactc gttccatgac gttcttgccc ttttcatcga aatgcggttg
     5581 aaacataatg gacaattgac atcctctttc agctggattc aaaggagtta aaattttaaa
     5641 cccaaatttg gagtttgatg tactggattg tggtatgtaa tacttggaat tcgtcaatag
     5701 atcctgtaaa aattgagtca aagcaacact tttttcacga agtttagata ctccacccac
     5761 tttagcatac acttccaatg acgacttcac agcaacaaca tcaagaacag aaggatttga
     5821 ctgtctgtaa gaaagagccg agtttattgg atcaaactct tctaacattt tgaatcgttc
     5881 ttgggagtta ttgccccacc aaccagctag tctaggaacg aaactgctct tcttgttctc
     5941 tatagtgtat ttttcatgca caaaaattcc acctatggct ccaggtcccg agtttaaata
     6001 tttatcgcgt gcattcagca attccaaaat cccattagag gggttcttca cgattatcta
     6061 tttgatcact ctcgcctaaa ctataaatat agctaccttt ttatcaatat acatgtactt
     6121 ccgaacatcg gcccttatct gttattaatc tattaataaa cccttaacaa tttcctgttg
     6181 ctccttgtaa accagtccta tatgtccaac ttgcccggat tggtctgatc ttccgatcta
     6241 acaatcacgt cttactaata ttataatgcc ttggtaacac gtctcgcgtt tacgcgtatg
     6301 ccctttagga attttgtaaa caaactgccc cgtagattac aaacaagaga gtgaattcca
     6361 ccctcaacta tagacctagg agccattttt ctatataact actatctgta taaggatggg
     6421 gcctcctggg acatgttcaa ttgaatttta cgcttcgaac catacacata acacgtaaaa
     6481 agctctaata ttgggcttct taaatggaaa gttgtagtat agattttacg cgatgatcca
     6541 attcgcgttt tgtgtaggga attgcttcta catcaagcaa aatttacaat gcttttatgt
     6601 atacggccgt agacggaata gacaacattg tataatcagg aattgatata aaatagttga
     6661 tatcccacta ttctaataac ttcatttttt attaattagt atttgataat ataaaatttc
     6721 attagttgct ttcaaacatt attcagacaa tctgtcatat ggataaaaag tatagtattg
     6781 gtttagatat tggtactaac tctgtgggtt gggcagttat cactgacgaa tataaagttc
     6841 catcaaagaa atttaaggtg ttaggtaaca ctgacagaca ctcaataaaa aagaatctta
     6901 tcggtgctct tttgttcgac tccggtgaaa ctgccgaggc tacacgttta aaaagaacag
     6961 caagaagaag atatacccgt agaaaaaata gaatatgtta tttacaagaa atcttttcta
     7021 atgaaatggc taaagttgat gattcctttt tccatagatt ggaagagtca tttttggttg
     7081 aagaagacaa aaagcatgag agacatccaa tctttgggaa tatagttgat gaagtggctt
     7141 accatgaaaa atatcctacc atttatcatt taagaaagaa attggtagat tcaactgata
     7201 aagctgacct tagattaatc tatttagcac ttgcccatat gattaaattt agaggtcatt
     7261 ttttgattga aggtgatttg aacccagata attctgacgt ggataaatta tttattcaat
     7321 tagtccaaac ctacaaccaa ttatttgagg aaaatccaat taatgctagt ggtgtcgatg
     7381 ccaaagctat attatcagcc agattatcaa aatctagacg tttggaaaat ttgattgccc
     7441 aattgccagg agaaaaaaag aatggattat ttggaaactt gatcgcatta tcattgggtt
     7501 tgacaccaaa ttttaaatct aattttgatt tagctgaaga tgctaaatta caattatcaa
     7561 aagacaccta tgacgacgat ttggacaatt tacttgctca aattggtgat caatatgcag
     7621 atttgttctt agctgctaaa aacttatctg atgctatttt gttgtctgat attttgagag
     7681 tgaacacaga aataaccaaa gctccattat cagcatctat gatcaaacgt tatgatgaac
     7741 accatcagga tttgacttta ttgaaagctt tggtgagaca acaattgcca gagaagtata
     7801 aagaaatctt tttcgatcaa tctaaaaacg ggtatgcagg ttatattgat gggggtgcct
     7861 cccaagagga attttacaaa tttataaaac ctattttaga aaagatggat gggactgagg
     7921 aacttttggt caaattgaac agagaagatt tgttacgtaa acagagaact tttgataatg
     7981 gtagtatacc tcaccaaatt catttgggtg agttgcatgc aattttaaga agacaagaag
     8041 atttttatcc atttttaaaa gataatagag aaaaaatcga gaaaatttta acctttagaa
     8101 ttccatacta tgttgggcct ttggctagag gtaattcaag atttgcctgg atgacacgta
     8161 aatcagaaga aactattacc ccttggaatt ttgaagaggt tgttgataaa ggagcatcag
     8221 cacagagttt tattgaaaga atgaccaatt tcgataaaaa cttaccaaat gaaaaagttt
     8281 taccaaaaca ttccttgtta tacgaatatt ttactgttta caatgaactt acaaaggtta
     8341 aatatgttac tgaaggtatg cgtaagccag cctttttatc tggagaacag aaaaaggcaa
     8401 tagttgattt attgtttaaa acaaatagaa aagttactgt taaacaatta aaagaagatt
     8461 actttaagaa aattgaatgt tttgattcag ttgaaatcag tggtgttgaa gacagattta
     8521 atgctagttt aggaacttac catgatttac ttaaaattat caaagataaa gatttcttgg
     8581 ataacgaaga aaatgaagac attttagaag acattgtttt aaccttaact ttattcgaag
     8641 atagagagat gattgaagaa cgtttgaaga cttatgcaca tttgtttgac gataaagtga
     8701 tgaaacagtt gaaaagaaga cgttatactg gatggggtag attgtctcgt aaattgatca
     8761 atggaattag agataaacaa agtggtaaaa ctatcttgga ctttttgaaa tctgacggat
     8821 ttgctaatag aaatttcatg caattgatcc acgacgatag tttgacattt aaagaagaca
     8881 tccaaaaggc ccaagtgagt gggcaaggtg attcattaca tgaacatatt gcaaatttag
     8941 ccggatctcc tgctattaag aaagggatat tacaaactgt taaagttgtg gatgaattag
     9001 tgaaagtaat gggaagacat aaacctgaaa acattgtcat tgagatggca agagaaaatc
     9061 aaactacaca aaaaggacag aaaaatagta gagaacgtat gaaaagaata gaagagggta
     9121 ttaaagaatt gggtagtcaa atattgaaag aacacccagt ggaaaatacc cagttgcaaa
     9181 atgaaaaatt atatctttac taccttcaaa atggacgtga tatgtatgtt gatcaggaat
     9241 tagatataaa tagactttca gattatgatg tagatcatat agttccacaa tctttcttga
     9301 aagatgattc catagacaat aaagtattaa ctagaagtga taaaaataga ggtaaaagtg
     9361 ataatgtccc aagtgaggaa gtcgtcaaaa agatgaaaaa ttactggcgt caacttttga
     9421 atgctaaatt aattactcaa agaaaatttg ataatttgac taaagcagaa agaggtgggc
     9481 tttctgaatt agataaagcc gggttcatta aaagacaatt ggtcgaaact agacaaatta
     9541 ctaaacatgt tgcccaaatt ttagattccc gtatgaacac taagtatgac gaaaatgata
     9601 agttaatacg tgaggttaaa gtcattactt taaaatcaaa acttgtctct gatttcagaa
     9661 aggatttcca attctataaa gttagagaaa ttaataatta tcatcatgct catgatgcat
     9721 atttgaatgc tgtagttgga actgctttaa tcaagaaata ccctaaatta gaatctgaat
     9781 ttgtatatgg tgattacaaa gtctatgatg ttagaaagat gattgctaaa tcagaacaag
     9841 aaattggtaa agctacagct aaatacttct tttactctaa cattatgaat ttctttaaaa
     9901 cagaaattac tttggcaaac ggtgaaatta gaaaaagacc tcttattgaa acaaatggtg
     9961 agactggaga gatagtttgg gacaaagggc gtgatttcgc tactgttaga aaagttttat
    10021 caatgccaca agttaacatt gtaaagaaaa cagaggttca aactggtggt ttctcaaaag
    10081 aaagtatttt gcctaaaaga aatagtgata aattgattgc cagaaaaaag gattgggatc
    10141 caaagaaata tggtggtttc gactcaccaa ccgtagccta ttctgttttg gttgtggcaa
    10201 aggttgaaaa gggtaaaagt aaaaagctta aatcagtaaa agaacttttg ggtattacaa
    10261 taatggaaag aagttccttt gaaaagaacc ctattgattt tttggaagct aaaggttata
    10321 aggaagtaaa gaaggactta ataatcaaat tgcctaaata ttctttattt gaattagaaa
    10381 atgggagaaa aagaatgttg gcttctgctg gagaattgca aaagggtaat gaattagcat
    10441 tgccttccaa atatgttaac ttcttgtatt tagcttcaca ctatgaaaag ttgaaagggt
    10501 caccagaaga taacgagcaa aaacaattat ttgttgaaca acacaaacac tacttagatg
    10561 agattataga acaaattagt gaattcagta aaagagtgat attagctgat gcaaatttag
    10621 ataaagtttt gtcagcctat aacaaacata gagataagcc aattagagaa caagcagaaa
    10681 acattattca cttatttacc cttaccaatt taggagcacc tgctgctttc aagtattttg
    10741 atacaacaat tgatcgtaaa agatatacct caacaaaaga agtcttagac gccaccttaa
    10801 ttcatcaatc aatcactgga ttgtatgaga caagaattga tttgtctcaa ttgggtggtg
    10861 atgaaggggc tgatcctaag aagaaaagaa aagttgatcc aaagaaaaag cgtaaggtgg
    10921 atcctaagaa aaagagaaag gttgactaca aagaccatga cggtgattat aaagatcatg
    10981 acatcgacta caaggatgac gatgacaagt gataatgact gcagagatcc atcgacctgc
    11041 cgccaagcta attccgggcg aatttctgtc gagtcatgta attagttatg tcacgcttac
    11101 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
    11161 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
    11221 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
    11281 tgcttgagaa ggttttggga cgctcgaaat cgcgtgcatt caacgctgca ggtcgacaac
    11341 ccttaatata acttggaaag caagcaatcg tagccccagc aaagcactat agtaggtgtt
    11401 tgctcttttt atttcctttg ctctacgcac tgtaatacag gattgatgaa agcagtatag
    11461 aaaatcgcct cgaagcctcg tgaatcagta gatctattta cattgttcgt ctcgtgcggt
    11521 ttttcaccag tgtcatgagc gagcgaactt ggtcgacatc attaaaaaaa actgcggctt
    11581 tcgtgtcaga gtcgctgtgt atcacttcga cgtaggtaat cagctcttaa gcttgaagtg
    11641 tggatcggaa ttttcttctc ctccttcatt agtcatcttc catgaaagag tgacttttga
    11701 ctcacacgaa ggaaaatgtg cgcaaattac aaatcaccta caagccacaa gacacattca
    11761 tcaaaggcac caaacaagac attcaagtgg accatgataa aagccaaggt ttgaaggcat
    11821 attctgtata cttcaatctt ggtttgctcg gcccaagtcc cggcgattag cggcgcccgg
    11881 tggcccggct ggatagattt caattggcac acgcacaatt atctccacaa ttgcattgca
    11941 tacaccttta caccacaact cggcgacctc ggccagcctc tgaaaacaca tagccaaaaa
    12001 cgaagaaggg acaggcattt tcccttcatt gcctagtatg gcataatacg tcaccaaacc
    12061 atgcattatg gattgttaag agctaatc
//
