LOCUS       sequence-360339-       10915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding PRDM9-Cas9 fusion under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10915)
  AUTHORS   Chen E, Lin-Shiao E, Trinidad M, Saffari Doost M, Colognori D, 
            Doudna JA
  TITLE     Decorating chromatin for enhanced genome editing using CRISPR-Cas9.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Dec 6;119(49):e2204259119. doi: 
            10.1073/pnas.2204259119. Epub 2022 Dec 2.
  PUBMED    36459645
REFERENCE   2  (bases 1 to 10915)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-12-6"; volume: "119(49):e2204259119. 
            doi"; pages: " 10.1073/pnas.2204259119. Epub 2022 Dec 2"
COMMENT     Sequence Label: PRDM9(KRAB-SSXRD-PR/SET-ZF1-dC)-Cas9
FEATURES             Location/Qualifiers
     source          1..10915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3032..7135
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3032..7135
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7139..7204
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7223..7243
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7250..7270
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7271..7327
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7328..7924
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7821..7841
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(7937..7956)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    8002..8057
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(8056..8075)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(8418..8434)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8418..8434)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8431..8453)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8442..8458
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(8466..8472,8473..8490,8491..8496))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8511..8532
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8591..8786
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      8637..8772
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    8792..8926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8829..8848)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8883..8902
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8994..9011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9165..9753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9245..9264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9924..10715,10716..10784))
                     /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     10547..10566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10785..10889)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccagggggg gsggggsggg gsgrggggsg
      541 gggsggggsg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgccaccat
     1741 gagccctgaa aagtcccaag aggagagccc agaagaagac acagagagaa cagagcggaa
     1801 gcccatggtc aaagatgcct tcaaagacat ttccatatac ttcaccaagg aagaatgggc
     1861 agagatggga gactgggaga aaactcgcta taggaatgtg aaaaggaact ataatgcact
     1921 gattactata ggtctcagag ccactcgacc agctttcatg tgtcaccgaa ggcaggccat
     1981 caaactccag gtggatgaca cagaagattc tgatgaagaa tggaccccta ggcagcaagt
     2041 caaacctcct tggatggcct taagagtgga acagcgtaaa caccagaagg gaatgcccaa
     2101 ggcgtcattc agtaatgaat ctagtttgaa agaattgtca agaacagcaa atttactgaa
     2161 tgcaagtggc tcagagcagg ctcagaaacc agtgtcccct tctggagaag caagtacctc
     2221 tggacagcac tcaagactaa aactggaact caggaagaag gagactgaaa gaaagatgta
     2281 tagcctgcga gaaagaaagg gtcatgcata caaagaggtc agcgagccgc aggatgatga
     2341 ttacctctat tgtgagatgt gtcagaactt cttcattgac agctgtgctg cccatgggcc
     2401 ccctacattt gtaaaggaca gtgcagtgga caaggggcac cccaaccgtt cagccctcag
     2461 tctgccccca gggctgagaa ttgggccatc aggcatccct caggctgggc ttggagtatg
     2521 gaatgaggca tctgatctgc cgctgggtct gcactttggc ccttatgagg gccgaattac
     2581 agaagacgaa gaggcagcca acaatggata ctcctggctg atcaccaagg ggagaaactg
     2641 ctatgagtat gtggatggaa aagataaatc ctgggccaac tggatgaggt atgtgaactg
     2701 tgcccgggat gatgaagagc agaacctggt ggccttccag taccacaggc agatcttcta
     2761 tagaacctgc cgagtcatta ggccaggctg tgaactgctg gtctggtatg gggatgaata
     2821 cggccaggaa ctgggcatca agtggggcag caagtggaag aaagagctca tggcagggag
     2881 agaaccaaag ccagagatcc atccatgtcc ctcatgctgt ctggcctttt caagtcagaa
     2941 atttctcagt caacatgtag aacgcaatca ctcctctcag aacttcggtg gtagtggagg
     3001 ttcaggagga tccgggggga gcggagggag catggacaag aaatacagca tcggcctgga
     3061 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
     3121 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggcgc
     3181 cctgctgttc gacagcggag aaacagccga ggccacccgg ctgaagagaa ccgccagaag
     3241 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
     3301 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
     3361 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     3421 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     3481 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     3541 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     3601 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
     3661 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     3721 cggcgagaag aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc
     3781 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     3841 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     3901 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     3961 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     4021 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     4081 tttcttcgac cagagcaaga acggctacgc cggctacatc gatggcggag ccagccagga
     4141 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     4201 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
     4261 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     4321 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     4381 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     4441 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgcca gcgcccagag
     4501 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     4561 gcacagcctg ctgtacgagt acttcaccgt gtacaacgag ctgaccaaag tgaaatacgt
     4621 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaaag ccatcgtgga
     4681 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     4741 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     4801 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     4861 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     4921 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     4981 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     5041 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     5101 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     5161 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     5221 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     5281 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     5341 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     5401 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     5461 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     5521 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     5581 ctccatcgat aacaaagtgc tgactcggag cgacaagaac cggggcaaga gcgacaacgt
     5641 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cgccagctgc tgaatgccaa
     5701 gctgattacc cagaggaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     5761 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
     5821 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaacg acaaactgat
     5881 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     5941 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     6001 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     6061 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     6121 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     6181 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaacagg
     6241 cgagatcgtg tgggataagg gccgggactt tgccaccgtg cggaaagtgc tgtctatgcc
     6301 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     6361 cctgcccaag aggaacagcg acaagctgat cgccagaaag aaggactggg accctaagaa
     6421 gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     6481 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     6541 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     6601 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     6661 gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc
     6721 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     6781 ggataatgag cagaaacagc tgtttgtgga acagcacaaa cactacctgg acgagatcat
     6841 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaggt
     6901 gctgagcgcc tacaacaagc acagagacaa gcctatcaga gagcaggccg agaatatcat
     6961 ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac
     7021 catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca
     7081 gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacgccga
     7141 ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag acgatgacga
     7201 taagagcctg ggcagcggct cccccaagaa aaaacgcaag gtggaagatc ctaagaaaaa
     7261 gcggaaagtg gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc
     7321 tggaccgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     7381 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     7441 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     7501 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     7561 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     7621 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     7681 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     7741 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     7801 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     7861 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     7921 ctgataagct agcagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     7981 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     8041 attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga
     8101 atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag
     8161 gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc
     8221 atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt
     8281 ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct
     8341 gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca
     8401 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     8461 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     8521 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     8581 cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     8641 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     8701 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     8761 tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta
     8821 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     8881 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga
     8941 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     9001 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     9061 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     9121 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     9181 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     9241 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     9301 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     9361 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     9421 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     9481 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     9541 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     9601 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     9661 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     9721 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     9781 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     9841 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     9901 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     9961 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    10021 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
    10081 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    10141 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    10201 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
    10261 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    10321 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    10381 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    10441 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    10501 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    10561 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    10621 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    10681 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    10741 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    10801 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
    10861 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctg
//
