LOCUS       sequence-244096-        9440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Streptococcus pyogenes Cas9 (SpCas9) and two gRNAs in 
            Leishmania.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9440)
  AUTHORS   Zhang WW, Lypaczewski P, Matlashewski G
  TITLE     Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania.
  JOURNAL   Methods Mol Biol. 2020;2116:199-224. doi: 
            10.1007/978-1-0716-0294-2_14.
  PUBMED    32221923
REFERENCE   2  (bases 1 to 9440)
  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"; journalName: "Methods Mol
            Biol."; date: "2020"; volume: "2116:199-224. doi"; pages: " 
            10.1007/978-1-0716-0294-2_14"
COMMENT     Sequence Label: pLdCN2
FEATURES             Location/Qualifiers
     source          1..9440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        60..135
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           142..209
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     ncRNA           222..258
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        281..356
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           363..430
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     regulatory      598..607
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             607..672
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             679..699
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             724..4824
                     /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             724..4824
                     /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             4825..4872
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4900..4917)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             6109..6903
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHKAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6163..6182)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6773..6792
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6929..6948)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6930..6948)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7035..7052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7206..7794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7286..7305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7965..8756,8757..8825))
                     /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     8588..8607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8826..8930)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8998..9016
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9054..9076)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9176..9195
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        9276..9294
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     9276..9293
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ttatcggggg ctcggttgag tttttggttt ggtgattttg tgggtcttcg agaagacctg
       61 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
      121 gcaccgagtc ggtgcttttt tggccggcat ggtcccagcc tcctcgctgg cgccggctgg
      181 gcaacatgct tcggcatggc gaatgggacg gatctcacca tctgatgagt ccgtgaggac
      241 gaaacgagta agctcgtcat ggtggggtct tcgagaagac gttttagagc tagaaatagc
      301 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      361 ttggccggca tggtcccagc ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg
      421 cgaatgggac ggatccccgg gtaccgagct ctctcttctc cttcccctct ctctcctctc
      481 tcctctctcc agagctctct cttctccttc ccctctctct cctctctcct ctctccagag
      541 ctcgaattcg caagcttagc acctgcctga aatcactttt tttcaggttg gaccggtgcc
      601 accatggact ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac
      661 gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
      721 gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
      781 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
      841 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
      901 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
      961 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     1021 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     1081 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     1141 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     1201 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     1261 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     1321 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     1381 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     1441 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     1501 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     1561 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     1621 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     1681 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     1741 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     1801 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     1861 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     1921 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     1981 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     2041 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     2101 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     2161 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     2221 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     2281 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     2341 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     2401 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     2461 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     2521 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     2581 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     2641 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     2701 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     2761 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     2821 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     2881 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     2941 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     3001 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     3061 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     3121 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     3181 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     3241 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     3301 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     3361 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     3421 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     3481 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     3541 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     3601 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     3661 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     3721 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     3781 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     3841 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     3901 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     3961 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     4021 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     4081 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     4141 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     4201 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     4261 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     4321 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     4381 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     4441 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     4501 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     4561 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     4621 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     4681 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     4741 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     4801 gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca
     4861 aaaaagaaaa agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     4921 gccagccatc tgttggatcc gcggccgcct cgagcccggg cgccgtcgac cgctgcttgc
     4981 gttcgcatgg acgccacgtt cgacccgatc gggcagcagg ccgcggagac gcggcagcgc
     5041 aaacgtgccc ggcgggtggc tcgttccgga ggaggcgttc cgccgtcgct acgtcagcat
     5101 gcaagaggat cattgctttg cggttgaggt ggcggagctc aactaccagc gtgccgtgga
     5161 atacgttcat caagcgtcag ccgccggcgc ggtcgcgaca cctgctcggc ggagcggcca
     5221 ctgtgctgct ctcttgacgg ccggtcgcga cgcagggccg ctggtgggtc tccagcgggt
     5281 acggtgcgat caggcagcca tgacggctac gctgagcacg cttgcgaagg agctggctgc
     5341 actgtcgccg tagcgcgatg cgagacgtat cgagcgcatg ccagtggggt gctgggcaac
     5401 acttggcggc gcgtggctct ttctcactgg tttgggcacg cccacgctgt gggtgtgtcg
     5461 gtgtctgccc tgccgctctc tctcctgatc tctcgtcctg gcaacaacaa cacaccgcga
     5521 atgagttgcc cacaagccat ctgcagtccg cacgcgcgtc cgggccttgc ggtgtggatg
     5581 cgagcatcgc gcattgccgg ccaccgccgc gtgccaaaag cacacacatc gctccccctc
     5641 acgtggtgag cgtctgcgcg tgtgtgtctg tgtgcataaa tgcgtacgtg tgcgctcggt
     5701 ccacggtcac catcgcgccc cgcggcatcg gtgctcgcac ctttccattc tctccgtacg
     5761 gcgttcgcca ctctgctaac tcttctcccc tttcctctct ccgcatctgc gccttgtgtg
     5821 ccgtgtgtgc gccccacacg tactctacac gccttctctg catgcgctcc tcgcctgcct
     5881 gaaacgatga cgtgcgccgc cactcgctca cccgctccca tccacatgca ttcctgcacc
     5941 ctacagcgac tctaagccgt acattccatc tcctcaacac ccactcgctt ccttctgctc
     6001 tcacctatta cttcgccagc ccacatatct gctataacct gccctccccc acccgcttcc
     6061 cacacatccg ccaccgctac gaagcctcgg taccgagctc gaattcgcat gattgaacaa
     6121 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     6181 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     6241 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     6301 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     6361 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     6421 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     6481 acgcttgatc cggctacctg cccattcgac cacaaagcga aacatcgcat cgagcgagca
     6541 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     6601 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     6661 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     6721 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     6781 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     6841 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     6901 tgatcgatga tatcagatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     6961 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     7021 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     7081 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     7141 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     7201 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     7261 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     7321 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     7381 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     7441 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     7501 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     7561 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     7621 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     7681 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     7741 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     7801 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     7861 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7921 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7981 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     8041 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     8101 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     8161 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     8221 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     8281 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     8341 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     8401 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     8461 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     8521 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     8581 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     8641 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     8701 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     8761 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     8821 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     8881 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8941 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     9001 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     9061 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     9121 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     9181 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     9241 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta tagaactcga
     9301 gtgtgagtta tgaggtctgc gattgacgta ggagttgcaa gggggagggg gtatgaacgg
     9361 ggtgggtaga gcttttttag gtggaagtag tgagagggtg ggcttgtgag aatttgaggt
     9421 gtgttcgtga tgtgtggatc
//
