LOCUS       sequence-347030-       11636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pNOC episomal plasmid harboring the dead version of humanized spCas9
            gene sequence tagged with Nlux and sgRNA targeting the fluorescent 
            gene tdtomato with spacer 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11636)
  AUTHORS   Naduthodi MIS, Sudfeld C, Avitzigiannis EK, Trevisan N, van Lith E, 
            Alcaide Sancho J, D'Adamo S, Barbosa M, van der Oost J
  TITLE     Comprehensive Genome Engineering Toolbox for Microalgae 
            Nannochloropsis oceanica Based on CRISPR-Cas Systems.
  JOURNAL   ACS Synth Biol. 2021 Dec 17;10(12):3369-3378. doi: 
            10.1021/acssynbio.1c00329. Epub 2021 Nov 18.
  PUBMED    34793143
REFERENCE   2  (bases 1 to 11636)
  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.1c00329"; journalName: "ACS Synth Biol"; date: 
            "2021-12-17- 17"; volume: "10"; issue: "12"; pages: "3369-3378"
COMMENT     Sequence Label: pNOC_dCas9_sgRNA Td1
FEATURES             Location/Qualifiers
     source          1..11636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     ncRNA           complement(1481..1548)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(1553..1628)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           complement(1649..1685)
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     CDS             2667..2678
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2899..2919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2920..2943,2944..2946,2947..5430,5431..5436,5437..5502,
                     5503..5505,5506..7020)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2920..2943,2944..2946,2947..5433,5434..5436,
                     5437..7020)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2920..7020
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7573..7599
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      7601..7610
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7615..7635
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     rep_origin      complement(9824..10412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9904..9923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10583..11374,11375..11443))
                     /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     11206..11225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11444..11548)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(11585..11636,1..452)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 aattaaaaat agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga
       61 tgtaaaagac tctaggggga tcgccaacaa atactacctt ttatcttgta cttcctgctc
      121 tcaggtatta atgccgaatt gtttcatctt gtctgtgtag aagaccacac acgaaaatcc
      181 tgtgatttta cattttactt atcgttaatc gaatgtatat ctatttaatc tgcttttctt
      241 gtctaataaa tatatatgta aagtacgctt tttgttgaaa ttttttaaac ctttgtttat
      301 ttttttttct tcattccgta actcttctac cttctttatt tactttctaa aatccaaata
      361 caaaacataa aaataaataa acacagagta aattcccaaa ttattccatc attaaaagat
      421 acgaggcgcg tgtaagttac aggcaagcga tccgtctaag aaaccggtgc aaagctgacg
      481 cccttttcgt cgtcgttgct ggtgttaaaa ttgcgcgttt cttttttttc cgtctttgtc
      541 gtcttttgac ttttttatct tcttttctgt cttcttttat tcttctcttc ttcttcatca
      601 ctccctcttc atttttcttc ttctttttcc tcgtctattg tcctcgggca ttaccagttg
      661 tgctgctcac gtgcgcgcaa gcggtgaaag ggtgagagga tacacggaca acaatcattg
      721 aaaattacgc ttaaagccgg tgcctggaaa agtccacgcg cagatggcct ttgcttgcct
      781 tgctgctgct gcagctgctg ctgctgtcgc ctgtgatggt aatggttgtt gttgtggtcc
      841 tgcttgcttg ctagtttgca ttgcgtttgc ggttgcctta tgaccgactc cgtgtgattg
      901 atccttaaac atattttatc attgatgtgg attgtgtgac gacggcaaaa aagaattact
      961 ttacacatga ctatcacacc tgctcccctc ccttctcctt tcaatcggtc aacttcccaa
     1021 ccgctcctcg aaaatatttc agttgaatcc tctactacgt accccttact taaatttact
     1081 ttctctctcc ccatcaatgc gccgttgttc tgaagttgta ttattgatgg gagatgattg
     1141 gcactgcacc agacctggcc aaaagccacc acaggtggcc acatactctt gcatgggtat
     1201 actctaccga ggcaaagatt aaagtgcacg tcccctgccc ctctctctcc ctacttcctt
     1261 cagtctcagt ggttccgtgc ccgttcagca ctacatcgcc ctgcccgctg ttccttctca
     1321 aacagccacc catcccaacc cacatacaca gaaacaacct ttcctacaca ttttcccaag
     1381 tcttctcttc actattctac actaatacac caccgtgata tgccggttat ctagatgtcc
     1441 tttctccttt ctcatccttt ctttcccatg gtacccagga gtcccattcg ccatgccgaa
     1501 gcatgttgcc cagccggcgc cagcgaggag gctgggacca tgccggccaa aagcaccgac
     1561 tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc
     1621 tctaaaactc atcaaagagt tcatgcgcga cgagcttact cgtttcgtcc tcacggactc
     1681 atcaggcgca tgctcttcat cgattctgtg gatggaggga gggaaggagg gggggagtga
     1741 gtgagtagag agaacgacga ctggggcaaa gagaggatga cgccactgca acaaaagaca
     1801 gggaagaata gaggctgttg gaaaacagga acacagtgaa gaatagaggc tgcagggtaa
     1861 caggcacatt tatcgcagag aggcgggcgg caggggcgat acgtaaagag acccctaaac
     1921 ttctccgcgt catccgttca cgctacctcc ttccctccct ttactcccat tgatcaccaa
     1981 ggcagcgccc gcagccacca gcagccaccg tcctcagtgt cacagctaga cgcgtcccca
     2041 tcaggcagca gaccaccaga atatgcgctt tcgcttccca tcatatccct ccctcctccc
     2101 cttctattcc tcatcatgca tcatttcttt ctccttgctg catgcccctc ttcttacctt
     2161 ggctaagatc taccggagga agaagtcttc aacgatagat aaatgcctgt atcgtgatgg
     2221 ttgtttgaat ggtggaagtg agccaggtat tgagaacgag gggcgcaagg gtgcgtggaa
     2281 gaaggcaggg agaggcgagg cgaaggagaa gggcatgtgg tgggaacgaa gcctccacac
     2341 cacagccacc catgcttttc tctgttcgag ttcccgggtt ctactctcac acactacacg
     2401 tgcacagaaa acattcacgc acaacagaga tacagaggga tccgtcgaga tggtctacac
     2461 gtcctaatgt cccctgcttt cgaagcccag agccatcgct ttctgttttt atccattcgc
     2521 aacacatcct gttctcgcga cctctcctct tcctggccac catcgcttct ccgtgccatg
     2581 ccctgtacat ccttccctcc ctcgttgtct accacactaa tcatgcgctc tgcagcagac
     2641 actcggctac tatttgtgtc gtacggattg aggggaggga ggggaaggga ggcaagaagt
     2701 aaatcggtgg tcaacaacag acgcggtctc cccaccctcc ctcattcctt tcccctccta
     2761 taatgagcga agaaagccat ggacaccgag tcacccactc tcctaatcca ccaccaccag
     2821 ggcactaacg gactcacgct caccaaactc ggtaacaata tccactgcac acagatacac
     2881 acgcaaccct cgagcatgcc caagaaaaag cggaaggtgg acaagaagta ctccattggg
     2941 ctcgctatcg gcacaaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg
     3001 agcaaaaaat tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt
     3061 ggcgccctcc tgttcgactc cggggagacg gccgaagcca cgcggctcaa aagaacagca
     3121 cggcgcagat atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat
     3181 gagatggcta aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag
     3241 gaggataaaa agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac
     3301 catgaaaagt acccaaccat atatcatctg aggaagaagc ttgtagacag tactgataag
     3361 gctgacttgc ggttgatcta tctcgcgctg gcgcatatga tcaaatttcg gggacacttc
     3421 ctcatcgagg gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg
     3481 gttcagactt acaatcagct tttcgaagag aacccgatca acgcatccgg agttgacgcc
     3541 aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag
     3601 ctccctgggg agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg
     3661 acccccaact ttaaatctaa cttcgacctg gccgaagatg ccaagcttca actgagcaaa
     3721 gacacctacg atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac
     3781 ctttttttgg cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg
     3841 aacacggaga tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac
     3901 caccaagact tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag
     3961 gaaattttct tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc
     4021 caggaggaat tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag
     4081 ctgctggtaa agcttaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga
     4141 agcatccccc accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat
     4201 ttctacccct ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata
     4261 ccctactatg taggccccct cgcccgggga aattccagat tcgcgtggat gactcgcaaa
     4321 tcagaagaga ccatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc
     4381 cagtccttca tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt
     4441 cctaaacact ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa
     4501 tacgtcacag aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc
     4561 gtggacctcc tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaagactat
     4621 ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac
     4681 gcatccctgg gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac
     4741 aatgaggaga acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat
     4801 agggagatga ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg
     4861 aaacagctca agaggcgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat
     4921 gggatccgag acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt
     4981 gccaaccgga acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc
     5041 cagaaagcac aagtttctgg ccagggggac agtcttcacg agcacatcgc taatcttgca
     5101 ggtagcccag ctatcaaaaa gggaatactg cagaccgtta aggtcgtgga tgaactcgtc
     5161 aaagtaatgg gaaggcataa gcccgagaat atcgttatcg agatggcccg agagaaccaa
     5221 actacccaga agggacagaa gaacagtagg gaaaggatga agaggattga agagggtata
     5281 aaagaactgg ggtcccaaat ccttaaggaa cacccagttg aaaacaccca gcttcagaat
     5341 gagaagctct acctgtacta cctgcagaac ggcagggaca tgtacgtgga tcaggaactg
     5401 gacatcaatc ggctctccga ctacgacgtg gatgctatcg tgccccagtc ttttctcaaa
     5461 gatgattcta ttgataataa agtgttgaca agatccgata aaaatagagg gaagagtgat
     5521 aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt attggcggca gctgctgaac
     5581 gccaaactga tcacacaacg gaagttcgat aatctgacta aggctgaacg aggtggcctg
     5641 tctgagttgg ataaagccgg cttcatcaaa aggcagcttg ttgagacacg ccagatcacc
     5701 aagcacgtgg cccaaattct cgattcacgc atgaacacca agtacgatga aaatgacaaa
     5761 ctgattcgag aggtgaaagt tattactctg aagtctaagc tggtctcaga tttcagaaag
     5821 gactttcagt tttataaggt gagagagatc aacaattacc accatgcgca tgatgcctac
     5881 ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc ccaagcttga atctgaattt
     5941 gtttacggag actataaagt gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa
     6001 ataggcaagg ccaccgctaa gtacttcttt tacagcaata ttatgaattt tttcaagacc
     6061 gagattacac tggccaatgg agagattcgg aagcgaccac ttatcgaaac aaacggagaa
     6121 acaggagaaa tcgtgtggga caagggtagg gatttcgcga cagtccggaa ggtcctgtcc
     6181 atgccgcagg tgaacatcgt taaaaagacc gaagtacaga ccggaggctt ctccaaggaa
     6241 agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc
     6301 aagaaatacg gcggattcga ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa
     6361 gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg aactgctggg catcacaatc
     6421 atggagcgat caagcttcga aaaaaacccc atcgactttc tcgaggcgaa aggatataaa
     6481 gaggtcaaaa aagacctcat cattaagctt cccaagtact ctctctttga gcttgaaaac
     6541 ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga aaggtaacga gctggcactg
     6601 ccctctaaat acgttaattt cttgtatctg gccagccact atgaaaagct caaagggtct
     6661 cccgaagata atgagcagaa gcagctgttc gtggaacaac acaaacacta ccttgatgag
     6721 atcatcgagc aaataagcga attctccaaa agagtgatcc tcgccgacgc taacctcgat
     6781 aaggtgcttt ctgcttacaa taagcacagg gataagccca tcagggagca ggcagaaaac
     6841 attatccact tgtttactct gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac
     6901 accaccatag acagaaagcg gtacacctct acaaaggagg tcctggacgc cacactgatt
     6961 catcagtcaa ttacggggct ctatgaaaca agaatcgacc tctctcagct cggtggagac
     7021 agcagggctg acgggtccgg cagcgggtcc gttaacatgg tgtttactct cgaggacttc
     7081 gtgggcgact ggcggcagac agctggctat aacctcgacc aggtgctgga gcagggcggt
     7141 gtgtcctcgc ttttccagaa cctgggcgtc tccgtgaccc caatccagcg catcgtcctg
     7201 agcggcgaga acggcctgaa gatcgacatc cacgtcatca ttccctacga gggcctgagc
     7261 ggggaccaaa tgggccagat cgagaagatc ttcaaggttg tgtaccccgt ggacgatcac
     7321 cacttcaagg tgatactgca ctacgggacg ctggtcatcg acggtgtgac gccgaacatg
     7381 attgactact tcggccgtcc ttacgagggc attgccgtgt tcgacggcaa gaagatcacg
     7441 gtgaccggca ccctgtggaa cggcaataag atcattgacg agcggctgat caacccggat
     7501 ggctcgctgt tgttccgcgt gaccatcaac ggggtaaccg gctggcgcct gtgcgagcgc
     7561 atcctggccg gataccccta cgacgtgccc gactacgcgt ccatggtggc tagcccgaaa
     7621 aagaagagga aggtctgaca attggaaaga tccaagagag acgagtagag attttttttt
     7681 tgggattgat gtttgtcgtt ctttgagttg tcgtcgagtt acgccttttg taagaatgtt
     7741 ccgcaggaga ggaggaggat gggcatgagt gagggtgaga gggcttgccc gctttttttt
     7801 ttaaaaacgc tgaagacgtg gttgtcaaac aaacccccca tagaaacgat tttgttacgg
     7861 tgcggtccag acgtcacttg aatggctccg cggaaaggcc agggagggaa ggggggaggg
     7921 aggaaacatg aaacatgttg aacggctcaa cagggtttgg gggacaagag aggtagcgcc
     7981 ctgatggact gctccctccc ctcctttccc tcaatgtctc attcatccat gcttccccct
     8041 tctctctctc ccctccgttc catcccccgc gggcgtggta gtggcgtgat gggatccact
     8101 aaaatgtacg tgtaagaaaa gccggtgagc ttacgctttt gtgaaagtgg gagtacgagt
     8161 gttgtgtgtg tgtgtagtgg tttcagaccc cagacagagg cgaagcagaa aaagcagacg
     8221 atgaagacga cgaagaaatg agcagtctat ttttatcgtg gaaacagaag aggtgatatc
     8281 gtctcctgca ggcatgcaag ctgatccact agaggccatg gcgatggagt ggatggagga
     8341 ggaggcgagc gtagcagcaa gcgtgagtta tacagccagg cacatgtcgc aatccttcgg
     8401 tctcgggctt gaaatccacg cactaatcac gctgggccat gcaaagagca atgccgaggc
     8461 ccaccacaca aaacgctgtg tcgcgcgttg cggcctgaag cttcatactt cttagtcgcc
     8521 gccaaaaggg ctcgagagac gagacccgtt ggcatgaccg atgttgttcg acgcggtttg
     8581 cttcgtcaca gtcgacgtga ttcaggaatc tggagcctgc agatcatttt tttcagcctg
     8641 atatcgttct tttccactga gaaccatcag accacctttt cttccattgt gtgaaggagt
     8701 aggagttgcc gtgctgcttt gtgggagaca tctgcgatgg tgaccagcct cccgtcgtct
     8761 ggtcgacgtg acgagcctct tcactgttct tcgacggaga gacgcaagcg agacggctct
     8821 agaccttttg gacacgcatt ctgtgtgtga actagtggac agtgatacca cgtctgaaag
     8881 ctcaccactg cccatggtgc agctacttgt cacaaagttt tgactccgtc ggtatcacca
     8941 ttcgcgctcg tgtgcctggt tgttccgcca cgccggcctg ccccggggcg gggcaatatt
     9001 ctaaaatctc acgcaaaaca ccgcacttac ccctcacaca tattcgtgat agaccaccac
     9061 caatctcagc ccgcatcaac acaggagggc ccatgcccct ctcgcaggag gagagcacgc
     9121 tgatcgagag ggctacggcg acgattaact cgattcctat ctccgaggat tactcggtgg
     9181 cttccgctgc tctgtcgtcc gacggacgga tcttcacggg ggtcaacgtg taccatttta
     9241 cgggggggcc ttgcgcggag ctggtggtcc tggggaccgc cgctgctgcc gcggctggga
     9301 acctgacgtg cattgtcgcc atcgggaacg agaaccgcgg gattctgagc ccctgcgggc
     9361 ggtgccggca ggtcctgctc gacctccacc ctgggatcaa ggcgattgtg aaggattccg
     9421 acgggcagcc tacggcggtc ggtatcagag aacttctccc ttcgggctac gtgtgggaga
     9481 gcgcttgaac tagtagtaga tgccgaccgg agtccgcaaa aatcaccagt ctctctctac
     9541 aaatctatct ctctctattt ttctccagaa taatgtgtga gtagttccca gataagggaa
     9601 ttagggttct tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat
     9661 ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca aaatccagtg
     9721 acctggttta aaccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     9781 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     9841 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     9901 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     9961 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    10021 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    10081 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    10141 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    10201 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    10261 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    10321 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    10381 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    10441 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    10501 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    10561 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    10621 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    10681 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    10741 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    10801 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    10861 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    10921 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    10981 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    11041 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    11101 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    11161 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    11221 atagcagaac tttaaaagtg ctcatcattg gaaaccgttc ttcggggcga aaactctcaa
    11281 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    11341 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    11401 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    11461 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    11521 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtcct
    11581 tttcatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa tatata
//
