LOCUS       sequence-245311-       12197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of KRAB-dCas9-BFP-KRAB from the AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12197)
  AUTHORS   Tian R, Gachechiladze MA, Ludwig CH, Laurie MT, Hong JY, Nathaniel 
            D, Prabhu AV, Fernandopulle MS, Patel R, Abshari M, Ward ME, 
            Kampmann M
  TITLE     CRISPR Interference-Based Platform for Multimodal Genetic Screens in
            Human iPSC-Derived Neurons.
  JOURNAL   Neuron. 2019 Aug 5. pii: S0896-6273(19)30640-3. doi: 
            10.1016/j.neuron.2019.07.014.
  PUBMED    31422865
REFERENCE   2  (bases 1 to 12197)
  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: "Neuron. 
            2019 Aug 5. pii: S0896-6273(19)30640-3. doi: 
            10.1016/j.neuron.2019.07.014."
COMMENT     Sequence Label: pMTL3
FEATURES             Location/Qualifiers
     source          1..12197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..804
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    complement(806..839)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    846..871
                     /label=SA
                     /note="splice acceptor site"
     CDS             895..948
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             958..1557
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(958..977)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1454..1474
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1589..1606)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1595..1819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(1823..1856)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        1859..2238
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2240..2517
                     /label=chicken beta-actin promoter
     intron          2519..3536
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3460..3482
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3544..3563
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3595..3604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3631..3816
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             join(3838..3864,3865..3867,3868..6354,6355..6357,
                     6358..7941)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3838..7941
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7945..7971
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7951..7977
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7990..8010
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8017..8037
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8077..8772
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             8818..9003
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     primer_bind     complement(9098..9117)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    9163..9218
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(9217..9236)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(9567..9600)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    9641..10477
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     rep_origin      complement(10525..11113)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10605..10624)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11284..12075,12076..12144))
                     /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     11907..11926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
       61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
      121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
      181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
      241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
      301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
      361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
      421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
      481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
      541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
      601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
      661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
      721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
      781 gtcccctcca ccccacagtg gggcaataac ttcgtatagc atacattata cgaagttata
      841 agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag cggagagggc
      901 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gctcgagatg
      961 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1021 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1081 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1141 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     1201 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     1261 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1321 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     1381 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     1441 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     1501 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgatct
     1561 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1621 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1681 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1741 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1801 gatgcggtgg gctctatggg tcataacttc gtatagcata cattatacga agttattcga
     1861 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     1921 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1981 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     2041 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     2101 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     2161 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     2221 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     2281 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     2341 ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
     2401 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
     2461 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgggg
     2521 agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     2581 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     2641 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
     2701 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
     2761 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
     2821 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     2881 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     2941 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     3001 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     3061 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     3121 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     3181 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     3241 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     3301 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     3361 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     3421 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     3481 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     3541 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaatgt cgacgccacc
     3601 atggatgcta agtcactaac tgcctggtcc cggacactgg tgaccttcaa ggatgtattt
     3661 gtggacttca ccagggagga gtggaagctg ctggacactg ctcagcagat cgtgtacaga
     3721 aatgtgatgc tggagaacta taagaacctg gtttccttgg gttatcagct tactaagcca
     3781 gatgtgatcc tccggttgga gaagggagaa gagcccggcg gttccggcgg agggtcgatg
     3841 gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc
     3901 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     3961 agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggagaaac agccgaggcc
     4021 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     4081 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     4141 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     4201 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     4261 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     4321 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     4381 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     4441 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     4501 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg
     4561 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     4621 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     4681 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     4741 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     4801 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     4861 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     4921 tacatcgatg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     4981 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     5041 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     5101 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     5161 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     5221 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     5281 gtggacaagg gcgccagcgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     5341 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtac
     5401 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     5461 ggcgagcaga aaaaagccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     5521 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     5581 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     5641 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     5701 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     5761 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     5821 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     5881 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     5941 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     6001 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     6061 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     6121 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     6181 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     6241 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     6301 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc
     6361 gtgcctcaga gctttctgaa ggacgactcc atcgataaca aagtgctgac tcggagcgac
     6421 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     6481 tactggcgcc agctgctgaa tgccaagctg attacccaga ggaagttcga caatctgacc
     6541 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     6601 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     6661 aagtacgacg agaacgacaa actgatccgg gaagtgaaag tgatcaccct gaagtccaag
     6721 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     6781 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     6841 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     6901 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     6961 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     7021 ctgatcgaga caaacggcga aacaggcgag atcgtgtggg ataagggccg ggactttgcc
     7081 accgtgcgga aagtgctgtc tatgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     7141 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgacaa gctgatcgcc
     7201 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     7261 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     7321 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     7381 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     7441 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     7501 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     7561 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     7621 cacaaacact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     7681 ctggccgacg ctaatctgga caaggtgctg agcgcctaca acaagcacag agacaagcct
     7741 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     7801 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     7861 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     7921 ctgtctcagc tgggaggcga cgcctatccc tatgacgtgc ccgattatgc cagcctgggc
     7981 agcggctccc ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg gaaagtggac
     8041 ggcattggta gtgggagcaa cggcagcagc ggatccagcg agctgattaa ggagaacatg
     8101 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
     8161 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     8221 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
     8281 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
     8341 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     8401 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
     8461 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc
     8521 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
     8581 ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
     8641 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc
     8701 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
     8761 cacaagctta atggcggtgg cggagggatg gatgctaagt cactaactgc ctggtcccgg
     8821 acactggtga ccttcaagga tgtatttgtg gacttcacca gggaggagtg gaagctgctg
     8881 gacactgctc agcagatcgt gtacagaaat gtgatgctgg agaactataa gaacctggtt
     8941 tccttgggtt atcagcttac taagccagat gtgatcctcc ggttggagaa gggagaagag
     9001 ccctagtaac gcgtgaattc actcctcagg tgcaggctgc ctatcagaag gtggtggctg
     9061 gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt
     9121 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     9181 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     9241 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     9301 tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc
     9361 ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat
     9421 tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag
     9481 ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga
     9541 gatccctcga cctgcagccc aagcttataa cttcgtatag catacattat acgaagttat
     9601 gcggccgcac cggtatgcat ttaattaaga tatcagcttt actagggaca ggattggtga
     9661 cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg gtctaacccc
     9721 cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg gagccatctc
     9781 tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat cctgggaggg
     9841 agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg ttctcagtgg
     9901 ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt ctggtgcgtt
     9961 tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca gtttggaaaa
    10021 acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt tctagtcccc
    10081 aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag tagccagccc
    10141 cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc ctttgtgaga
    10201 atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc tctttctcca
    10261 tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg cccagagcag
    10321 ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg gcaagcccag
    10381 gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc ccctggctct
    10441 cctgcccctt ccctacaggg gttcctggct ctgctctaag ggcgaattcg ccaggaaccg
    10501 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
    10561 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
    10621 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
    10681 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
    10741 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
    10801 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
    10861 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
    10921 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
    10981 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
    11041 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
    11101 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
    11161 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
    11221 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
    11281 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
    11341 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
    11401 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
    11461 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
    11521 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
    11581 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
    11641 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
    11701 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
    11761 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
    11821 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
    11881 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
    11941 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
    12001 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
    12061 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
    12121 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
    12181 gggttgaatt cgccctt
//
