LOCUS       sequence-202045-       13701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9-Nlux and sgRNA for Nitrate Reductase target 1. 
            Hygromycin resistance and contains CEN/ARS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13701)
  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 13701)
  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-ARS-CRISPR-sgNR2
FEATURES             Location/Qualifiers
     source          1..13701
                     /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"
     misc_feature    439..942
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     ncRNA           complement(2165..2232)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(2237..2312)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           complement(2333..2369)
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     CDS             3344..3355
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3576..3596
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3597..7697
                     /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             3597..7697
                     /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             3597..7697
                     /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             8250..8276
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      8278..8287
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8292..8312
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      8993..9002
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9832..10857
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             9832..10857
                     /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(11093..11110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11264..11852)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11344..11363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12023..12814,12815..12883))
                     /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     12646..12665
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12884..12988)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(13066..13521)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13203..13224)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     13415..13434
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13647..13669
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     13661..13678
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     13662..13678
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(13685..13701,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(13685..13701,1..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 gaccggtggc
      421 cggccggttt cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat
      481 gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt
      541 ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag
      601 gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca
      661 gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt
      721 gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag
      781 agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa
      841 acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt tatatattta
      901 tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtgtt
      961 taaacaccgg tcgaggaggc attgtattta cagcggagag cgaagaatga agtgagagga
     1021 agttaggagg gaggaaaaga caaagacggg ctgcattaag agaaagtgaa gagcatcagc
     1081 agcgcatcac agaaaatgat tgagggaaca acatcttcaa atgtatgaaa caaaaaggat
     1141 ggaagtggaa gagcaaagct gacgcccttt tcgtcgtcgt tgctggtgtt aaaattgcgc
     1201 gtttcttttt tttccgtctt tgtcgtcttt tgactttttt atcttctttt ctgtcttctt
     1261 ttattcttct cttcttcttc atcactccct cttcattttt cttcttcttt ttcctcgtct
     1321 attgtcctcg ggcattacca gttgtgctgc tcacgtgcgc gcaagcggtg aaagggtgag
     1381 aggatacacg gacaacaatc attgaaaatt acgcttaaag ccggtgcctg gaaaagtcca
     1441 cgcgcagatg gcctttgctt gccttgctgc tgctgcagct gctgctgctg tcgcctgtga
     1501 tggtaatggt tgttgttgtg gtcctgcttg cttgctagtt tgcattgcgt ttgcggttgc
     1561 cttatgaccg actccgtgtg attgatcctt aaacatattt tatcattgat gtggattgtg
     1621 tgacgacggc aaaaaagaat tactttacac atgactatca cacctgctcc cctcccttct
     1681 cctttcaatc ggtcaacttc ccaaccgctc ctcgaaaata tttcagttga atcctctact
     1741 acgtacccct tacttaaatt tactttctct ctccccatca atgcgccgtt gttctgaagt
     1801 tgtattattg atgggagatg attggcactg caccagacct ggccaaaagc caccacaggt
     1861 ggccacatac tcttgcatgg gtatactcta ccgaggcaaa gattaaagtg cacgtcccct
     1921 gcccctctct ctccctactt ccttcagtct cagtggttcc gtgcccgttc agcactacat
     1981 cgccctgccc gctgttcctt ctcaaacagc cacccatccc aacccacata cacagaaaca
     2041 acctttccta cacattttcc caagtcttct cttcactatt ctacactaat acaccaccgt
     2101 gatatgccgg ttatctagat gtcctttctc ctttctcatc ctttctttcc catggtaccc
     2161 aggagtccca ttcgccatgc cgaagcatgt tgcccagccg gcgccagcga ggaggctggg
     2221 accatgccgg ccaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
     2281 ttattttaac ttgctatttc tagctctaaa acccagtaca gacgcgcgag acgacgagct
     2341 tactcgtttc gtcctcacgg actcatcagg tctcgatcga ttctgtggat ggagggaggg
     2401 aaggaggggg ggagtgagtg agtcgagaga acgacgactg gggcaaagag aggatgacgc
     2461 cactgcaaca aaagacaggg aagaatagag gctgttggaa aacaggaaca cagtgaagaa
     2521 tagaggctgc agggtaacag gcacatttat cgcagagagg cgggcggcag gggcgatacg
     2581 taaagagacc cctaaacttc tccgcgtcat ccgttcacgc tacctccttc cctcccttta
     2641 ctcccattga tcaccaaggc agcgcccgca gccaccagca gccaccgtcc tcagtgtcac
     2701 agctagacgc gtccccatca ggcagcagac caccagaata tgcgctttcg cttcccatca
     2761 tatccctccc tcctcccctt ctattcctca tcatgcatca tttctttctc cttgctgcat
     2821 gcccctcttc ttaccttggc taagatctac cggaggaaga agtcttcaac gatagataaa
     2881 tgcctgtatc gtgatggttg tttgaatggt ggaagtgagc caggtattga gaacgagggg
     2941 cgcaagggtg cgtggaagaa ggcagggaga ggcgaggcga aggagaaggg catgtggtgg
     3001 gaacgaagcc tccacaccac agccacccat gcttttctct gttcgagttc ccgggttcta
     3061 ctctcacaca ctacacgtgc acagaaaaca ttcacgcaca acagagatac agagggatcc
     3121 gtcgagatgg tctacacgtc ctaatgtccc ctgctttcga agcccagagc catcgctttc
     3181 tgtttttatc cattcgcaac acatcctgtt ctcgcgacct ctcctcttcc tggccaccat
     3241 cgcttctccg tgccatgccc tgtacatcct tccctccctc gttgtctacc acactaatca
     3301 tgcgctctgc agcagacact cggctactat ttgtgtcgta cggattgagg ggagggaggg
     3361 gaagggaggc aagaagtaaa tcggtggtca acaacagacg cggtctcccc accctccctc
     3421 attcctttcc cctcctataa tgagcgaaga aagccatgga caccgagtca cccactctcc
     3481 taatccacca ccaccagggc actaacggac tcacgctcac caaactcggt aacaatatcc
     3541 actgcacaca gatacacacg caaccctcga gcatgcccaa gaaaaagcgg aaggtggaca
     3601 agaagtactc cattgggctc gatatcggca caaacagcgt cggctgggcc gtcattacgg
     3661 acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat cgccacagca
     3721 taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc gaagccacgc
     3781 ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc tgctacctgc
     3841 aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat aggctggagg
     3901 agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt ggcaatatcg
     3961 tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg aagaagcttg
     4021 tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg catatgatca
     4081 aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc gatgtcgaca
     4141 aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac ccgatcaacg
     4201 catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc cggcggctcg
     4261 aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt aatcttatcg
     4321 ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc gaagatgcca
     4381 agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg gcccagatcg
     4441 gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc attctgctga
     4501 gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct agtatgatca
     4561 agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc agacagcaac
     4621 tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac gccggataca
     4681 ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc ttggaaaaaa
     4741 tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg cgcaaacagc
     4801 gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg cacgctatcc
     4861 tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag attgagaaaa
     4921 tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat tccagattcg
     4981 cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag gaagtcgtgg
     5041 ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat aaaaatctgc
     5101 ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca gtttataacg
     5161 agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc ctgtctggag
     5221 agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt accgtgaaac
     5281 agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa atcagcggag
     5341 tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa atcattaaag
     5401 acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt gtcctcaccc
     5461 ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac gctcatctct
     5521 tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg gggcggctgt
     5581 caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc ctggattttc
     5641 ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat gactctctca
     5701 cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt cttcacgagc
     5761 acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag accgttaagg
     5821 tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc gttatcgaga
     5881 tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa aggatgaaga
     5941 ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac ccagttgaaa
     6001 acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc agggacatgt
     6061 acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat catatcgtgc
     6121 cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga tccgataaaa
     6181 atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg aaaaattatt
     6241 ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat ctgactaagg
     6301 ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg cagcttgttg
     6361 agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg aacaccaagt
     6421 acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag tctaagctgg
     6481 tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac aattaccacc
     6541 atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa aaatatccca
     6601 agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg aaaatgatcg
     6661 caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac agcaatatta
     6721 tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag cgaccactta
     6781 tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag
     6841 tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg
     6901 gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg atcgcacgca
     6961 aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc gcttacagtg
     7021 tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac
     7081 tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg
     7141 aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc aagtactctc
     7201 tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag
     7261 gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc agccactatg
     7321 aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg gaacaacaca
     7381 aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg
     7441 ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat aagcccatca
     7501 gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc gcgcctgcag
     7561 ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca aaggaggtcc
     7621 tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga atcgacctct
     7681 ctcagctcgg tggagacagc agggctgacg ggtccggcag cgggtccgtt aacatggtgt
     7741 ttactctcga ggacttcgtg ggcgactggc ggcagacagc tggctataac ctcgaccagg
     7801 tgctggagca gggcggtgtg tcctcgcttt tccagaacct gggcgtctcc gtgaccccaa
     7861 tccagcgcat cgtcctgagc ggcgagaacg gcctgaagat cgacatccac gtcatcattc
     7921 cctacgaggg cctgagcggg gaccaaatgg gccagatcga gaagatcttc aaggttgtgt
     7981 accccgtgga cgatcaccac ttcaaggtga tactgcacta cgggacgctg gtcatcgacg
     8041 gtgtgacgcc gaacatgatt gactacttcg gccgtcctta cgagggcatt gccgtgttcg
     8101 acggcaagaa gatcacggtg accggcaccc tgtggaacgg caataagatc attgacgagc
     8161 ggctgatcaa cccggatggc tcgctgttgt tccgcgtgac catcaacggg gtaaccggct
     8221 ggcgcctgtg cgagcgcatc ctggccggat acccctacga cgtgcccgac tacgcgtcca
     8281 tggtggctag cccgaaaaag aagaggaagg tctgacaatt ggaaagatcc aagagagacg
     8341 agtagagatt ttttttttgg gattgatgtt tgtcgttctt tgagttgtcg tcgagttacg
     8401 ccttttgtaa gaatgttccg caggagagga ggaggatggg catgagtgag ggtgagaggg
     8461 cttgcccgct ttttttttta aaaacgctga agacgtggtt gtcaaacaaa ccccccatag
     8521 aaacgatttt gttacggtgc ggtccagacg tcacttgaat ggctccgcgg aaaggccagg
     8581 gagggaaggg gggagggagg aaacatgaaa catgttgaac ggctcaacag ggtttggggg
     8641 acaagagagg tagcgccctg atggactgct ccctcccctc ctttccctca atgtctcatt
     8701 catccatgct tcccccttct ctctctcccc tccgttccat cccccgcggg cgtggtagtg
     8761 gcgtgatggg atccactaaa atgtacgtgt aagaaaagcc ggtgagctta cgcttttgtg
     8821 aaagtgggag tacgagtgtt gtgtgtgtgt gtagtggttt cagaccccag acagaggcga
     8881 agcagaaaaa gcagacgatg aagacgacga agaaatgagc agtctatttt tatcgtggaa
     8941 acagaagagg tgatatcgtc tcctgcaggc atgcaagctg atccactaga ggccatggcg
     9001 gccgcactag tggatccccc gggatgcata cttggcctag gaagatggag tggatggagg
     9061 aggaggcgag cgtagcagca agcgtgagtt atacagccag gcacatgtcg caatccttcg
     9121 gtctcgggct taaaatccac gcactaatca cgctgggcca tgcaaagagc aatgccgagg
     9181 cccaccacac aaaacgctgt gtcgcgcgtt gcggcctgaa gcttcatact tcttagtcgc
     9241 cgccaaaagg gctcgagaga cgagacccgt tggcatgacc gatgttgttc gacgcggttt
     9301 gcttcgtcac agtcgacgtg attcaggaat ctggagcctg cagatcattt ttttcagcct
     9361 gatatcgttc ttttccactg agaaccatca gaccaccttt tcttccattg tgtgaaggag
     9421 taggagttgc cgtgctgctt tgtgggagac atctgcgatg gtgaccagcc tcccgtcgtc
     9481 tggtcgacgt gacgagcctc ttcactgttc ttcgacggag agacgcaagc gagacggctc
     9541 tagacctttt ggacacgcat tctgtgtgtg aactagtgga cagtgatacc acgtctgaaa
     9601 gctcaccact gcccatggtg cagctacttg tcacaaagtt ttgactccgt cggtatcacc
     9661 attcgcgctc gtgtgcctgg ttgttccgcc acgccggcct gccccggggc ggggcaatat
     9721 tctaaaatct cacgcaaaac accgcactta cccctcacac atattcgtga tagaccacca
     9781 ccaatctcag cccgcatcaa cacagtcgag ggccccgggg ggcaataaga tatgaaaaag
     9841 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
     9901 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     9961 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
    10021 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
    10081 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
    10141 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
    10201 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
    10261 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
    10321 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
    10381 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
    10441 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
    10501 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
    10561 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
    10621 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
    10681 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
    10741 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
    10801 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagagt
    10861 agatgccgac cggagtccgc aaaaatcacc agtctctctc tacaaatcta tctctctcta
    10921 tttttctcca gaataatgtg tgagtagttc ccagataagg gaattagggt tcttataggg
    10981 tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac
    11041 ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtgaccagct tcctcgctca
    11101 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
    11161 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
    11221 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
    11281 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
    11341 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
    11401 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    11461 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    11521 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    11581 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    11641 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    11701 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    11761 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    11821 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    11881 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    11941 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    12001 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    12061 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    12121 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    12181 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    12241 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    12301 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    12361 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    12421 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    12481 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    12541 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    12601 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    12661 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    12721 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    12781 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    12841 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    12901 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    12961 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg
    13021 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
    13081 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
    13141 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
    13201 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
    13261 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
    13321 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
    13381 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
    13441 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
    13501 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg
    13561 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
    13621 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
    13681 attgtaatac gactcactat a
//
