LOCUS       sequence-202006-       13078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9-GFP and sgRNA in Nannochloropsis oceanica, contains 
            hygromycin resistance cassette, for integration into geneome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13078)
  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 13078)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..13078
                     /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             join(6952..6954,6955..6957,6958..7668)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(6997..7018)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     7605..7626
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7669..7689
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      8370..8379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9209..10234
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             9209..10234
                     /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(10470..10487)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10641..11229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10721..10740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11400..12191,12192..12260))
                     /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     12023..12042
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12261..12365)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(12443..12898)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12580..12601)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12792..12811
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13024..13046
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     13038..13055
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     13039..13055
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(13062..13078,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(13062..13078,1..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 catggtgagc
     6961 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     7021 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     7081 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     7141 accttcacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     7201 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     7261 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     7321 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     7381 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     7441 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     7501 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     7561 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     7621 ttcgtgaccg ccgccgggat cactcacggc atggacgagc tgtacaagcc gaaaaagaag
     7681 aggaaggtct gacaattgga aagatccaag agagacgagt agagattttt tttttgggat
     7741 tgatgtttgt cgttctttga gttgtcgtcg agttacgcct tttgtaagaa tgttccgcag
     7801 gagaggagga ggatgggcat gagtgagggt gagagggctt gcccgctttt ttttttaaaa
     7861 acgctgaaga cgtggttgtc aaacaaaccc cccatagaaa cgattttgtt acggtgcggt
     7921 ccagacgtca cttgaatggc tccgcggaaa ggccagggag ggaagggggg agggaggaaa
     7981 catgaaacat gttgaacggc tcaacagggt ttgggggaca agagaggtag cgccctgatg
     8041 gactgctccc tcccctcctt tccctcaatg tctcattcat ccatgcttcc cccttctctc
     8101 tctcccctcc gttccatccc ccgcgggcgt ggtagtggcg tgatgggatc cactaaaatg
     8161 tacgtgtaag aaaagccggt gagcttacgc ttttgtgaaa gtgggagtac gagtgttgtg
     8221 tgtgtgtgta gtggtttcag accccagaca gaggcgaagc agaaaaagca gacgatgaag
     8281 acgacgaaga aatgagcagt ctatttttat cgtggaaaca gaagaggtga tatcgtctcc
     8341 tgcaggcatg caagctgatc cactagaggc catggcggcc gcactagtgg atcccccggg
     8401 atgcatactt ggcctaggaa gatggagtgg atggaggagg aggcgagcgt agcagcaagc
     8461 gtgagttata cagccaggca catgtcgcaa tccttcggtc tcgggcttaa aatccacgca
     8521 ctaatcacgc tgggccatgc aaagagcaat gccgaggccc accacacaaa acgctgtgtc
     8581 gcgcgttgcg gcctgaagct tcatacttct tagtcgccgc caaaagggct cgagagacga
     8641 gacccgttgg catgaccgat gttgttcgac gcggtttgct tcgtcacagt cgacgtgatt
     8701 caggaatctg gagcctgcag atcatttttt tcagcctgat atcgttcttt tccactgaga
     8761 accatcagac caccttttct tccattgtgt gaaggagtag gagttgccgt gctgctttgt
     8821 gggagacatc tgcgatggtg accagcctcc cgtcgtctgg tcgacgtgac gagcctcttc
     8881 actgttcttc gacggagaga cgcaagcgag acggctctag accttttgga cacgcattct
     8941 gtgtgtgaac tagtggacag tgataccacg tctgaaagct caccactgcc catggtgcag
     9001 ctacttgtca caaagttttg actccgtcgg tatcaccatt cgcgctcgtg tgcctggttg
     9061 ttccgccacg ccggcctgcc ccggggcggg gcaatattct aaaatctcac gcaaaacacc
     9121 gcacttaccc ctcacacata ttcgtgatag accaccacca atctcagccc gcatcaacac
     9181 agtcgagggc cccggggggc aataagatat gaaaaagcct gaactcaccg cgacgtctgt
     9241 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     9301 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     9361 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     9421 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     9481 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     9541 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     9601 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     9661 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     9721 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     9781 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     9841 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     9901 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     9961 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
    10021 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
    10081 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
    10141 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
    10201 acgccccagc actcgtccga gggcaaagga atagagtaga tgccgaccgg agtccgcaaa
    10261 aatcaccagt ctctctctac aaatctatct ctctctattt ttctccagaa taatgtgtga
    10321 gtagttccca gataagggaa ttagggttct tatagggttt cgctcatgtg ttgagcatat
    10381 aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt
    10441 cctaaaacca aaatccagtg accagcttcc tcgctcactg actcgctgcg ctcggtcgtt
    10501 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
    10561 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
    10621 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
    10681 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
    10741 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
    10801 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
    10861 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
    10921 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
    10981 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
    11041 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
    11101 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
    11161 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
    11221 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
    11281 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
    11341 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
    11401 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
    11461 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
    11521 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
    11581 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
    11641 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
    11701 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
    11761 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
    11821 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
    11881 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
    11941 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
    12001 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
    12061 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
    12121 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
    12181 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
    12241 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
    12301 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
    12361 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
    12421 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
    12481 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
    12541 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
    12601 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
    12661 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
    12721 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
    12781 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
    12841 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
    12901 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
    12961 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
    13021 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactata
//
