LOCUS       sequence-416282-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR pooled KO of human LYSET.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Richards CM, Jabs S, Qiao W, Varanese LD, Schweizer M, Mosen PR, 
            Riley NM, Klussendorf M, Zengel JR, Flynn RA, Rustagi A, Widen JC, 
            Peters CE, Ooi YS, Xie X, Shi PY, Bartenschlager R, Puschnik AS, 
            Bogyo M, Bertozzi CR, Blish CA, Winter D, Nagamine CM, Braulke T, 
            Carette JE
  TITLE     The human disease gene LYSET is essential for lysosomal enzyme 
            transport and viral infection.
  JOURNAL   Science. 2022 Sep 8:eabn5648. doi: 10.1126/science.abn5648.
  PUBMED    36074821
REFERENCE   2  (bases 1 to 13013)
  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: "Science. 
            2022 Sep 8:eabn5648. doi: 10.1126/science.abn5648."
COMMENT     Sequence Label: plentiCRISPRv2-PURO-LYSET-gRNA1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1291..1665)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(1684..1731)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(1779..2108)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1793..1928
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1847..1866)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2093..2113
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2122..2550)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2232..2253)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2444..2463
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2596..2820
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2809..2826
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             2852..3085
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3157..3745
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3263..3274
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3672..3692
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3761..4357)
                     /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     complement(3844..3864)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(4358..4414)
                     /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             complement(4424..4447)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(4448..4495)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(4496..8599)
                     /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             complement(4496..8599)
                     /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"
     regulatory      8596..8605
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(8624..8835)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(8897..8972)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(8994..9242)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(9052..9071)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(9222..9242)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    9293..9410
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(9708..9752)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    9937..10170
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    10663..10788
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             10835..11015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(11033..11231)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(11058..11078)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        11233..11612
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     11784..11803
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        11878..11982
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11983..12051,12052..12843)
                     /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     complement(12201..12220)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca cgaagtgcac
     1321 gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct cgccgatctc
     1381 ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac acgacctccg accactcggc
     1441 gtacagctcg tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg
     1501 gtcctggacc gcgctgatga acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc
     1561 ctccacgaag tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac
     1621 gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg gccatggttt agttcctcac
     1681 cttgtcgtat tatactatgc cgatatacta tgccgatgat taattgtcaa cacgtgctga
     1741 tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc taggcctcca
     1801 aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc ggcctctgca
     1861 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     1921 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt
     1981 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
     2041 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac
     2101 tgacacacat tccacagaat taattcgcgt taaatttttg ttaaatcagc tcatttttta
     2161 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2221 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2281 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2341 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2401 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2461 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2521 ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc cctagagccc cagctggttc
     2581 tttccgcctc agaagccata gagcccaccg catccccagc atgcctgcta ttgtcttccc
     2641 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca
     2701 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca
     2761 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag
     2821 tcgaggctga tcagcgggtt taaacgggcc ctgctagaga ttttccacac tgactaaaag
     2881 ggtctgaggg atctctagtt accagagtca cacaacagac gggcacacac tacttgaagc
     2941 actcaaggca agctttattg aggcttaagc agtgggttcc ctagttagcc agagagctcc
     3001 caggctcaga tctggtctaa ccagagagac ccagtacaag caaaaagcag atcttgtctt
     3061 cgttgggagt gaattagccc ttccagtccc cccttttctt ttaaaaagtg gctaagatct
     3121 acagctgcct tgtaagtcat tggtcttaaa gtcgacgcgg ggaggcggcc caaagggaga
     3181 tccgactcgt ctgagggcga aggcgaagac gcggaagagg ccgcagagcc ggcagcaggc
     3241 cgcgggaagg aaggtccgct ggattgaggg ccgaagggac gtagcagaag gacgtcccgc
     3301 gcagaatcca ggtggcaaca caggcgagca gccaaggaaa ggacgatgat ttccccgaca
     3361 acaccacgga attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg
     3421 cggcgatgag ttccgccgtg gcaataggga gggggaaagc gaaagtcccg gaaaggagct
     3481 gacaggtggt ggcaatgccc caaccagtgg gggttgcgtc agcaaacaca gtgcacacca
     3541 cgccacgttg cctgacaacg ggccacaact cctcataaag agacagcaac caggatttat
     3601 acaaggagga gaaaatgaaa gccatacggg aagcaatagc atgatacaaa ggcattaaag
     3661 cagcgtatcc acatagcgta aaaggagcaa catagttaag aataccagtc aatctttcac
     3721 aaattttgta atccagaggt tgattgtcga cttaacgcgt tcaggcaccg ggcttgcggg
     3781 tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga
     3841 gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg
     3901 cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt
     3961 ggtcgggcga gactccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg
     4021 ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc
     4081 gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg
     4141 ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga
     4201 ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg acggtgtggc
     4261 gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt
     4321 cgcgggtggc gaggcgcacc gtgggcttgt actcggtcgg tccaggattc tcttcgacat
     4381 ctccggcttg tttcagcaga gagaagtttg ttgcgccgga tcccttatcg tcatcgtctt
     4441 tgtaatcttt cttcttctta gcctgtccag ccttctttgt ggcggcaggt cgcttgtcgc
     4501 ctcccagctg agacaggtcg atccgtgtct cgtacaggcc ggtgatgctc tggtggatca
     4561 gggtggcgtc cagcacctct ttggtgctgg tgtacctctt ccggtcgatg gtggtgtcaa
     4621 agtacttgaa ggcggcaggg gctcccagat tggtcagggt aaacaggtgg atgatattct
     4681 cggcctgctc tctgatgggc ttatcccggt gcttgttgta ggcggacagc actttgtcca
     4741 gattagcgtc ggccaggatc actctcttgg agaactcgct gatctgctcg atgatctcgt
     4801 ccaggtagtg cttgtgctgt tccacaaaca gctgtttctg ctcattatcc tcgggggagc
     4861 ccttcagctt ctcatagtgg ctggccaggt acaggaagtt cacatatttg gagggcaggg
     4921 ccagttcgtt tcccttctgc agttcgccgg cagaggccag cattctcttc cggccgtttt
     4981 ccagctcgaa cagggagtac ttaggcagct tgatgatcag gtcctttttc acttctttgt
     5041 agcccttggc ttccagaaag tcgatgggat tcttctcgaa gctgcttctt tccatgatgg
     5101 tgatccccag cagctctttc acactcttca gtttcttgga cttgcccttt tccactttgg
     5161 ccaccaccag cacagaatag gccacggtgg ggctgtcgaa gccgccgtac ttcttagggt
     5221 cccagtcctt ctttctggcg atcagcttat cgctgttcct cttgggcagg atagactctt
     5281 tgctgaagcc gcctgtctgc acctcggtct ttttcacgat attcacttgg ggcatgctca
     5341 gcactttccg cacggtggca aaatcccggc ccttatccca cacgatctcc ccggtttcgc
     5401 cgtttgtctc gatcagaggc cgcttccgga tctcgccgtt ggccagggta atctcggtct
     5461 tgaaaaagtt catgatgttg ctgtagaaga agtacttggc ggtagccttg ccgatttcct
     5521 gctcgctctt ggcgatcatc ttccgcacgt cgtacacctt gtagtcgccg tacacgaact
     5581 cgctttccag cttagggtac tttttgatca gggcggttcc cacgacggcg ttcaggtagg
     5641 cgtcgtgggc gtggtggtag ttgttgatct cgcgcacttt gtaaaactgg aaatccttcc
     5701 ggaaatcgga caccagcttg gacttcaggg tgatcacttt cacttcccgg atcagcttgt
     5761 cattctcgtc gtacttagtg ttcatccggg agtccaggat ctgtgccacg tgctttgtga
     5821 tctgccgggt ttccaccagc tgtctcttga tgaagccggc cttatccagt tcgctcaggc
     5881 cgcctctctc ggccttggtc agattgtcga actttctctg ggtaatcagc ttggcgttca
     5941 gcagctgccg ccagtagttc ttcatcttct tcacgacctc ttcggagggc acgttgtcgc
     6001 tcttgccccg gttcttgtcg cttctggtca gcaccttgtt gtcgatggag tcgtccttca
     6061 gaaagctctg aggcacgata tggtccacat cgtagtcgga cagccggttg atgtccagtt
     6121 cctggtccac gtacatatcc cgcccattct gcaggtagta caggtacagc ttctcgttct
     6181 gcagctgggt gttttccacg gggtgttctt tcaggatctg gctgcccagc tctttgatgc
     6241 cctcttcgat ccgcttcatt ctctcgcggc tgttcttctg tcccttctgg gtggtctggt
     6301 tctctctggc catttcgatc acgatgttct cgggcttgtg ccggcccatc actttcacga
     6361 gctcgtccac caccttcact gtctgcagga tgcccttctt aatggcgggg ctgccggcca
     6421 gattggcaat gtgctcgtgc aggctatcgc cctggccgga cacctgggct ttctggatgt
     6481 cctctttaaa ggtcaggctg tcgtcgtgga tcagctgcat gaagtttctg ttggcgaagc
     6541 cgtcggactt caggaaatcc aggattgtct tgccggactg cttgtcccgg atgccgttga
     6601 tcagcttccg gctcagcctg ccccagccgg tgtatctccg ccgcttcagc tgcttcatca
     6661 ctttgtcgtc gaacaggtgg gcataggttt tcagccgttc ctcgatcatc tctctgtcct
     6721 caaacagtgt cagggtcagc acgatatctt ccagaatgtc ctcgttttcc tcattgtcca
     6781 ggaagtcctt gtccttgata attttcagca gatcgtggta tgtgcccagg gaggcgttga
     6841 accgatcttc cacgccggag atttccacgg agtcgaagca ctcgattttc ttgaagtagt
     6901 cctctttcag ctgcttcacg gtcactttcc ggttggtctt gaacagcagg tccacgatgg
     6961 cctttttctg ctcgccgctc aggaaggcgg gctttctcat tccctcggtc acgtatttca
     7021 ctttggtcag ctcgttatac acggtgaagt actcgtacag caggctgtgc ttgggcagca
     7081 ccttctcgtt gggcaggttc ttatcgaagt tggtcatccg ctcgatgaag ctctgggcgg
     7141 aagcgccctt gtccaccact tcctcgaagt tccagggggt gatggtttcc tcgctctttc
     7201 tggtcatcca ggcgaatctg ctgtttcccc tggccagagg gcccacgtag taggggatgc
     7261 ggaaggtcag gatcttctcg atcttttccc ggttgtcctt caggaatggg taaaaatctt
     7321 cctgccgccg cagaatggcg tgcagctctc ccaggtggat ctggtggggg atgctgccgt
     7381 tgtcgaaggt ccgctgcttc cgcagcaggt cctctctgtt cagcttcacg agcagttcct
     7441 cggtgccgtc catcttttcc aggatgggct tgatgaactt gtagaactct tcctggctgg
     7501 ctccgccgtc aatgtagccg gcgtagccgt tcttgctctg gtcgaagaaa atctctttgt
     7561 acttctcagg cagctgctgc cgcacgagag ctttcagcag ggtcaggtcc tggtggtgct
     7621 cgtcgtatct cttgatcata gaggcgctca ggggggcctt ggtgatctcg gtgttcactc
     7681 tcaggatgtc gctcagcagg atggcgtcgg acaggttctt ggcggccaga aacaggtcgg
     7741 cgtactggtc gccgatctgg gccagcaggt tgtccaggtc gtcgtcgtag gtgtccttgc
     7801 tcagctgcag tttggcatcc tcggccaggt cgaagttgct cttgaagttg ggggtcaggc
     7861 ccaggctcag ggcaatcagg tttccgaaca ggccattctt cttctcgccg ggcagctggg
     7921 cgatcagatt ttccagccgt ctgctcttgc tcagtctggc agacaggatg gccttggcgt
     7981 ccacgccgct ggcgttgatg gggttttcct cgaacagctg gttgtaggtc tgcaccagct
     8041 ggatgaacag cttgtccacg tcgctgttgt cggggttcag gtcgccctcg atcaggaagt
     8101 ggccccggaa cttgatcatg tgggccaggg ccagatagat cagccgcagg tcggccttgt
     8161 cggtgctgtc caccagtttc tttctcaggt ggtagatggt ggggtacttc tcgtggtagg
     8221 ccacctcgtc cacgatgttg ccgaagatgg ggtgccgctc gtgcttctta tcctcttcca
     8281 ccaggaagga ctcttccagt ctgtggaaga agctgtcgtc caccttggcc atctcgttgc
     8341 tgaagatctc ttgcagatag cagatccggt tcttccgtct ggtgtatctt cttctggcgg
     8401 ttctcttcag ccgggtggcc tcggctgttt cgccgctgtc gaacagcagg gctccgatca
     8461 ggttcttctt gatgctgtgc cggtcggtgt tgcccagcac cttgaatttc ttgctgggca
     8521 ccttgtactc gtcggtgatc acggcccagc ccacagagtt ggtgccgatg tccaggccga
     8581 tgctgtactt cttgtccatg gtggcagcgc tctagaaccg gtcctgtgtt ctggcggcaa
     8641 acccgttgcg aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac
     8701 cctcgggaaa aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc
     8761 ttctctaggc accggatcaa ttgccgaccc ctccccccaa cttctcgggg actgtgggcg
     8821 atgtgcgctc tgcccactga cgggcaccgg agccaattcc cactcctttc aagacctagc
     8881 tagcgaattc aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
     8941 ttattttaac ttgctatttc tagctctaaa actgcctttt tgggtgtgga cacggtgttt
     9001 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     9061 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     9121 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
     9181 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     9241 tcggtacctt aattaaccaa actggatctc tgctgtccct gtaataaacc cgaaaatttt
     9301 gaatttttgt aatttgtttt tgtaattctt tagtttgtat gtctgttgct attatgtcta
     9361 ctattctttc ccctgcactg taccccccaa tccccccttt tcttttaaaa ttgtggatga
     9421 atactgccat ttgtctgcag aattggcgca cgcagtgccg atccgttcac taatcgaatg
     9481 gatctgtctc tgtctctctc tccaccttct tcttctattc cttcgggcct gtcgggtccc
     9541 ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat atccctgcct aactctattc
     9601 actatagaaa gtacagcaaa aactattctt aaacctacca agcctcctac tatcattatg
     9661 aataatttta tataccacag ccaatttgtt atgttaaacc aattccacaa acttgcccat
     9721 ttatctaatt ccaataattc ttgttcattc ttttcttgct ggttttgcga ttcttcaatt
     9781 aaggagtgta ttaagcttgt gtaattgtta atttctctgt cccactccat ccaggtcgtg
     9841 tgattccaaa tctgttccag agatttatta ctccaactag cattccaagg cacagcagtg
     9901 gtgcaaatga gttttccaga gcaaccccaa atccccagga gctgttgatc ctttaggtat
     9961 ctttccacag ccaggattct tgcctggagc tgcttgatgc cccagactgt gagttgcaac
    10021 agatgctgtt gcgcctcaat agccctcagc aaattgttct gctgctgcac tataccagac
    10081 aataattgtc tggcctgtac cgtcagcgtc attgacgctg cgcccatagt gcttcctgct
    10141 gctcccaaga acccaaggaa caaagctcct attcccactg ctcttttttc tctctgcacc
    10201 actcttctct ttgccttggt gggtgctact cctaatggtt caatttttac tactttatat
    10261 ttatataatt cacttctcca attgtccctc atatctcctc ctccaggtct gaagatcagc
    10321 ggccgcttgc tgtgcggtgg tcttactttt gttttgctct tcctctatct tgtctaaagc
    10381 ttccttggtg tcttttatct ctatcctttg atgcacacaa tagagggttg ctactgtatt
    10441 atataatgat ctaagttctt ctgatcctgt ctgaagggat ggttgtagct gtcccagtat
    10501 ttgtctacag ccttctgatg tttctaacag gccaggatta actgcgaatc gttctagctc
    10561 cctgcttgcc catactatat gttttaattt atattttttc tttccccctg gccttaaccg
    10621 aattttttcc catcgcgatc taattctccc ccgcttaata ctgacgctct cgcacccatc
    10681 tctctccttc tagcctccgc tagtcaaaat ttttggcgta ctcaccagtc gccgcccctc
    10741 gcctcttgcc gtgcgcgctt cagcaagccg agtcctgcgt cgagagagct cctctggttt
    10801 ccctttcgct ttcaagtccc tgttcgggcg ccactgctag agattttcca cactgactaa
    10861 aagggtctga gggatctcta gttaccagag tcacacaaca gacgggcaca cactacttga
    10921 agcactcaag gcaagcttta ttgaggctta agcagtgggt tccctagtta gccagagagc
    10981 tcccaggctc agatctggtc taaccagaga gacccagtac aggcaaaacg cgctgcttat
    11041 atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg
    11101 acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg
    11161 gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca
    11221 aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa
    11281 gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc
    11341 aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg
    11401 taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata
    11461 cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac
    11521 cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga
    11581 ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc gtacatcgcg
    11641 aagcagcgca aaacgcctaa ccctaagcag attcttcatg caattgtcgg tcaagccttg
    11701 ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca cacaagcagg
    11761 gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag
    11821 gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc
    11881 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
    11941 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
    12001 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
    12061 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
    12121 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
    12181 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
    12241 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
    12301 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
    12361 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
    12421 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
    12481 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
    12541 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
    12601 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
    12661 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
    12721 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
    12781 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
    12841 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
    12901 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
    12961 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
