LOCUS       sequence-182812-       13072 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 13072)
  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 13072)
  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..13072
                     /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             9201..10226
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             9201..10226
                     /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(10464..10481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10635..11223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10715..10734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11394..12185,12186..12254))
                     /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     12017..12036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12255..12359)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(12437..12892)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12574..12595)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12786..12805
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13018..13040
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     13032..13049
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     13033..13049
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(13056..13072,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(13056..13072,1..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 cacgcaacac 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 ggaagatgga gtggatggag gaggaggcga gcgtagcagc aagcgtgagt
     8461 tatacagcca ggcacatgtc gcaatccttc ggtctcgggc ttaaaatcca cgcactaatc
     8521 acgctgggcc atgcaaagag caatgccgag gcccaccaca caaaacgctg tgtcgcgcgt
     8581 tgcggcctga agcttcatac ttcttagtcg ccgccaaaag ggctcgagag acgagacccg
     8641 ttggcatgac cgatgttgtt cgacgcggtt tgcttcgtca cagtcgacgt gattcaggaa
     8701 tctggagcct gcagatcatt tttttcagcc tgatatcgtt cttttccact gagaaccatc
     8761 agaccacctt ttcttccatt gtgtgaagga gtaggagttg ccgtgctgct ttgtgggaga
     8821 catctgcgat ggtgaccagc ctcccgtcgt ctggtcgacg tgacgagcct cttcactgtt
     8881 cttcgacgga gagacgcaag cgagacggct ctagaccttt tggacacgca ttctgtgtgt
     8941 gaactagtgg acagtgatac cacgtctgaa agctcaccac tgcccatggt gcagctactt
     9001 gtcacaaagt tttgactccg tcggtatcac cattcgcgct cgtgtgcctg gttgttccgc
     9061 cacgccggcc tgccccgggg cggggcaata ttctaaaatc tcacgcaaaa caccgcactt
     9121 acccctcaca catattcgtg atagaccacc accaatctca gcccgcatca acacaggagg
     9181 gccccggggg gcaataagat atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     9241 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     9301 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     9361 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     9421 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     9481 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     9541 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     9601 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     9661 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     9721 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     9781 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     9841 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     9901 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     9961 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
    10021 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
    10081 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
    10141 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
    10201 gcactcgtcc gagggcaaag gaatagagta gatgccgacc ggagtccgca aaaatcacca
    10261 gtctctctct acaaatctat ctctctctat ttttctccag aataatgtgt gagtagttcc
    10321 cagataaggg aattagggtt cttatagggt ttcgctcatg tgttgagcat ataagaaacc
    10381 cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac
    10441 caaaatccag tgacctgagc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
    10501 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
    10561 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
    10621 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
    10681 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
    10741 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
    10801 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
    10861 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
    10921 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    10981 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    11041 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
    11101 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    11161 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    11221 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    11281 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
    11341 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
    11401 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
    11461 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
    11521 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
    11581 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
    11641 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
    11701 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
    11761 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
    11821 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
    11881 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
    11941 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
    12001 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
    12061 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
    12121 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    12181 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    12241 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    12301 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    12361 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
    12421 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
    12481 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
    12541 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
    12601 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
    12661 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
    12721 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
    12781 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
    12841 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
    12901 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
    12961 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
    13021 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta ta
//
