LOCUS       sequence-201983-       12925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9-Nlux and sgRNA in Nannochloropsis oceanica, contains 
            hygromycin resistance cassette, for integration into genome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12925)
  AUTHORS   Poliner E, Takeuchi T, Du ZY, Benning C, Farre EM
  TITLE     Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR 
            System in the Oleaginous Microalga, Nannochloropsis oceanica 
            CCMP1779.
  JOURNAL   ACS Synth Biol. 2018 Apr 20;7(4):962-968. doi: 
            10.1021/acssynbio.7b00362. Epub 2018 Mar 14.
  PUBMED    29518315
REFERENCE   2  (bases 1 to 12925)
  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"; doi: 
            "10.1021/acssynbio.7b00362"; journalName: "ACS Synth Biol"; date: 
            "2018-04-20- 20"; volume: "7"; issue: "4"; pages: "962-968"
COMMENT     Sequence Label: pNOC-CRISPR
FEATURES             Location/Qualifiers
     source          1..12925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           complement(1430..1497)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(1502..1577)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2568..2579
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2800..2820
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2821..6921
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2821..6921
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2821..6921
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7474..7500
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      7502..7511
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7516..7536
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      8217..8226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9056..10081
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             9056..10081
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(10317..10334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10488..11076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10568..10587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11247..12038,12039..12107))
                     /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     11870..11889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12108..12212)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(12290..12745)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12427..12448)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12639..12658
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12871..12893
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12885..12902
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12886..12902
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(12909..12925,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(12909..12925,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt
      121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc
      181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt
      241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg
      301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga
      361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgca
      421 aagctgacgc ccttttcgtc gtcgttgctg gtgttaaaat tgcgcgtttc ttttttttcc
      481 gtctttgtcg tcttttgact tttttatctt cttttctgtc ttcttttatt cttctcttct
      541 tcttcatcac tccctcttca tttttcttct tctttttcct cgtctattgt cctcgggcat
      601 taccagttgt gctgctcacg tgcgcgcaag cggtgaaagg gtgagaggat acacggacaa
      661 caatcattga aaattacgct taaagccggt gcctggaaaa gtccacgcgc agatggcctt
      721 tgcttgcctt gctgctgctg cagctgctgc tgctgtcgcc tgtgatggta atggttgttg
      781 ttgtggtcct gcttgcttgc tagtttgcat tgcgtttgcg gttgccttat gaccgactcc
      841 gtgtgattga tccttaaaca tattttatca ttgatgtgga ttgtgtgacg acggcaaaaa
      901 agaattactt tacacatgac tatcacacct gctcccctcc cttctccttt caatcggtca
      961 acttcccaac cgctcctcga aaatatttca gttgaatcct ctactacgta ccccttactt
     1021 aaatttactt tctctctccc catcaatgcg ccgttgttct gaagttgtat tattgatggg
     1081 agatgattgg cactgcacca gacctggcca aaagccacca caggtggcca catactcttg
     1141 catgggtata ctctaccgag gcaaagatta aagtgcacgt cccctgcccc tctctctccc
     1201 tacttccttc agtctcagtg gttccgtgcc cgttcagcac tacatcgccc tgcccgctgt
     1261 tccttctcaa acagccaccc atcccaaccc acatacacag aaacaacctt tcctacacat
     1321 tttcccaagt cttctcttca ctattctaca ctaatacacc accgtgatat gccggttatc
     1381 tagatgtcct ttctcctttc tcatcctttc tttcccatgg tacccaggag tcccattcgc
     1441 catgccgaag catgttgccc agccggcgcc agcgaggagg ctgggaccat gccggccaaa
     1501 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct
     1561 atttctagct ctaaaacgaa gagccaaggc ctgctcttca tcgattctgt ggatggaggg
     1621 agggaaggag ggggggagtg agtgagtcga gagaacgacg actggggcaa agagaggatg
     1681 acgccactgc aacaaaagac agggaagaat agaggctgtt ggaaaacagg aacacagtga
     1741 agaatagagg ctgcagggta acaggcacat ttatcgcaga gaggcgggcg gcaggggcga
     1801 tacgtaaaga gacccctaaa cttctccgcg tcatccgttc acgctacctc cttccctccc
     1861 tttactccca ttgatcacca aggcagcgcc cgcagccacc agcagccacc gtcctcagtg
     1921 tcacagctag acgcgtcccc atcaggcagc agaccaccag aatatgcgct ttcgcttccc
     1981 atcatatccc tccctcctcc ccttctattc ctcatcatgc atcatttctt tctccttgct
     2041 gcatgcccct cttcttacct tggctaagat ctaccggagg aagaagtctt caacgataga
     2101 taaatgcctg tatcgtgatg gttgtttgaa tggtggaagt gagccaggta ttgagaacga
     2161 ggggcgcaag ggtgcgtgga agaaggcagg gagaggcgag gcgaaggaga agggcatgtg
     2221 gtgggaacga agcctccaca ccacagccac ccatgctttt ctctgttcga gttcccgggt
     2281 tctactctca cacactacac gtgcacagaa aacattcacg cacaacagag atacagaggg
     2341 atccgtcgag atggtctaca cgtcctaatg tcccctgctt tcgaagccca gagccatcgc
     2401 tttctgtttt tatccattcg caacacatcc tgttctcgcg acctctcctc ttcctggcca
     2461 ccatcgcttc tccgtgccat gccctgtaca tccttccctc cctcgttgtc taccacacta
     2521 atcatgcgct ctgcagcaga cactcggcta ctatttgtgt cgtacggatt gaggggaggg
     2581 aggggaaggg aggcaagaag taaatcggtg gtcaacaaca gacgcggtct ccccaccctc
     2641 cctcattcct ttcccctcct ataatgagcg aagaaagcca tggacaccga gtcacccact
     2701 ctcctaatcc accaccacca gggcactaac ggactcacgc tcaccaaact cggtaacaat
     2761 atccactgca cacagataca cacgcaaccc tcgagcatgc ccaagaaaaa gcggaaggtg
     2821 gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg ggccgtcatt
     2881 acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac
     2941 agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc
     3001 acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac
     3061 ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg
     3121 gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat
     3181 atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag
     3241 cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg
     3301 atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc
     3361 gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc
     3421 aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg
     3481 ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt
     3541 atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat
     3601 gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag
     3661 atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg
     3721 ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg
     3781 atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag
     3841 caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga
     3901 tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa
     3961 aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa
     4021 cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct
     4081 atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag
     4141 aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga
     4201 ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc
     4261 gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat
     4321 ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat
     4381 aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct
     4441 ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg
     4501 aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc
     4561 ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt
     4621 aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc
     4681 acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat
     4741 ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg
     4801 ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat
     4861 tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct
     4921 ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac
     4981 gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt
     5041 aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc
     5101 gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg
     5161 aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt
     5221 gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac
     5281 atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc
     5341 gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat
     5401 aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat
     5461 tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact
     5521 aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt
     5581 gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc
     5641 aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag
     5701 ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac
     5761 caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat
     5821 cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg
     5881 atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat
     5941 attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca
     6001 cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg
     6061 acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag
     6121 accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca
     6181 cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac
     6241 agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag
     6301 gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt
     6361 ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac
     6421 tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag
     6481 aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac
     6541 tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa
     6601 cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc
     6661 ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc
     6721 atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct
     6781 gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag
     6841 gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac
     6901 ctctctcagc tcggtggaga cagcagggct gacgggtccg gcagcgggtc cgttaacatg
     6961 gtgtttactc tcgaggactt cgtgggcgac tggcggcaga cagctggcta taacctcgac
     7021 caggtgctgg agcagggcgg tgtgtcctcg cttttccaga acctgggcgt ctccgtgacc
     7081 ccaatccagc gcatcgtcct gagcggcgag aacggcctga agatcgacat ccacgtcatc
     7141 attccctacg agggcctgag cggggaccaa atgggccaga tcgagaagat cttcaaggtt
     7201 gtgtaccccg tggacgatca ccacttcaag gtgatactgc actacgggac gctggtcatc
     7261 gacggtgtga cgccgaacat gattgactac ttcggccgtc cttacgaggg cattgccgtg
     7321 ttcgacggca agaagatcac ggtgaccggc accctgtgga acggcaataa gatcattgac
     7381 gagcggctga tcaacccgga tggctcgctg ttgttccgcg tgaccatcaa cggggtaacc
     7441 ggctggcgcc tgtgcgagcg catcctggcc ggatacccct acgacgtgcc cgactacgcg
     7501 tccatggtgg ctagcccgaa aaagaagagg aaggtctgac aattggaaag atccaagaga
     7561 gacgagtaga gatttttttt ttgggattga tgtttgtcgt tctttgagtt gtcgtcgagt
     7621 tacgcctttt gtaagaatgt tccgcaggag aggaggagga tgggcatgag tgagggtgag
     7681 agggcttgcc cgcttttttt tttaaaaacg ctgaagacgt ggttgtcaaa caaacccccc
     7741 atagaaacga ttttgttacg gtgcggtcca gacgtcactt gaatggctcc gcggaaaggc
     7801 cagggaggga aggggggagg gaggaaacat gaaacatgtt gaacggctca acagggtttg
     7861 ggggacaaga gaggtagcgc cctgatggac tgctccctcc cctcctttcc ctcaatgtct
     7921 cattcatcca tgcttccccc ttctctctct cccctccgtt ccatcccccg cgggcgtggt
     7981 agtggcgtga tgggatccac taaaatgtac gtgtaagaaa agccggtgag cttacgcttt
     8041 tgtgaaagtg ggagtacgag tgttgtgtgt gtgtgtagtg gtttcagacc ccagacagag
     8101 gcgaagcaga aaaagcagac gatgaagacg acgaagaaat gagcagtcta tttttatcgt
     8161 ggaaacagaa gaggtgatat cgtctcctgc aggcatgcaa gctgatccac tagaggccat
     8221 ggcggccgca ctagtggatc ccccgggatg catacttggc ctaggaagat ggagtggatg
     8281 gaggaggagg cgagcgtagc agcaagcgtg agttatacag ccaggcacat gtcgcaatcc
     8341 ttcggtctcg ggcttaaaat ccacgcacta atcacgctgg gccatgcaaa gagcaatgcc
     8401 gaggcccacc acacaaaacg ctgtgtcgcg cgttgcggcc tgaagcttca tacttcttag
     8461 tcgccgccaa aagggctcga gagacgagac ccgttggcat gaccgatgtt gttcgacgcg
     8521 gtttgcttcg tcacagtcga cgtgattcag gaatctggag cctgcagatc atttttttca
     8581 gcctgatatc gttcttttcc actgagaacc atcagaccac cttttcttcc attgtgtgaa
     8641 ggagtaggag ttgccgtgct gctttgtggg agacatctgc gatggtgacc agcctcccgt
     8701 cgtctggtcg acgtgacgag cctcttcact gttcttcgac ggagagacgc aagcgagacg
     8761 gctctagacc ttttggacac gcattctgtg tgtgaactag tggacagtga taccacgtct
     8821 gaaagctcac cactgcccat ggtgcagcta cttgtcacaa agttttgact ccgtcggtat
     8881 caccattcgc gctcgtgtgc ctggttgttc cgccacgccg gcctgccccg gggcggggca
     8941 atattctaaa atctcacgca aaacaccgca cttacccctc acacatattc gtgatagacc
     9001 accaccaatc tcagcccgca tcaacacagt cgagggcccc ggggggcaat aagatatgaa
     9061 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     9121 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     9181 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     9241 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     9301 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     9361 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     9421 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     9481 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     9541 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     9601 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     9661 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     9721 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     9781 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     9841 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     9901 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     9961 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
    10021 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
    10081 gagtagatgc cgaccggagt ccgcaaaaat caccagtctc tctctacaaa tctatctctc
    10141 tctatttttc tccagaataa tgtgtgagta gttcccagat aagggaatta gggttcttat
    10201 agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa
    10261 atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtgacc agcttcctcg
    10321 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
    10381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
    10441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
    10501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
    10561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
    10621 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
    10681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    10741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    10801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    10861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    10921 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    10981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    11041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    11101 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    11161 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    11221 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    11281 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    11341 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    11401 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    11461 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    11521 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    11581 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    11641 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    11701 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    11761 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    11821 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    11881 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    11941 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    12001 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    12061 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    12121 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    12181 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat
    12241 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
    12301 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
    12361 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
    12421 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
    12481 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
    12541 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
    12601 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
    12661 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
    12721 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
    12781 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
    12841 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
    12901 gtgaattgta atacgactca ctata
//
