LOCUS       sequence-244570-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting mouse Mlst8 gene by CRISPR-Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Hwang Y, Kim LC, Song W, Edwards DN, Cook RS, Chen J
  TITLE     Disruption of the Scaffolding Function of mLST8 Selectively Inhibits
            mTORC2 Assembly and Function and Suppresses mTORC2-Dependent Tumor 
            Growth In Vivo.
  JOURNAL   Cancer Res. 2019 Jul 1;79(13):3178-3184. doi: 
            10.1158/0008-5472.CAN-18-3658. Epub 2019 May 13.
  PUBMED    31085701
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"; doi: 
            "10.1158/0008-5472.CAN-18-3658"; journalName: "Cancer Res"; date: 
            "2019-07-1- 1"; volume: "79"; issue: "13"; pages: "3178-3184"
COMMENT     Sequence Label: sgMlst8_2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    290..407
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        458..706
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     458..478
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     629..648
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        728..803
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        865..1076
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1095..1104
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1101..5204
                     /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             1101..5204
                     /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             5205..5252
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5253..5276
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5286..5342
                     /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             5343..5939
                     /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     5836..5856
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5955..6543
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6008..6028)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6426..6437)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6615..6848
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6874..6891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6880..7104
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7150..7578
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7237..7256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7447..7468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7587..7607)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7592..7921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7772..7907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7834..7853
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7969..8016
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8035..8409
                     /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"
     polyA_signal    8539..8660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8576..8595)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8630..8649
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8709..8725)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8709..8725)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8722..8744)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8733..8749
                     /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(8757..8763,8764..8781,8782..8787))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8802..8823
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8940..8957)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9111..9699)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9191..9210)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9870..10661,10662..10730))
                     /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     10493..10512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10731..10835)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10910..10929)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11101..11480
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11482..11680
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11635..11655
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11698..11878
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11925..12050
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12543..12776
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12961..13005
                     /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"
ORIGIN
        1 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
       61 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
      121 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg
      181 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
      241 ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa
      301 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
      361 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
      421 gggacagcag agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc
      481 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
      541 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      601 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      661 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtat gtctggaacc
      721 tgacagggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      781 aaaaagtggc accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg
      841 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
      901 tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg
      961 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     1021 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc
     1081 ggttctagag cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca
     1141 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     1201 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     1261 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     1321 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     1381 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     1441 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     1501 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     1561 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     1621 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     1681 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     1741 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     1801 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     1861 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     1921 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     1981 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     2041 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     2101 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     2161 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     2221 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     2281 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     2341 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     2401 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     2461 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     2521 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     2581 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     2641 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     2701 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     2761 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     2821 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     2881 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     2941 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     3001 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     3061 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     3121 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     3181 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     3241 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     3301 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     3361 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     3421 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     3481 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     3541 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     3601 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     3661 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     3721 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     3781 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     3841 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     3901 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     3961 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     4021 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     4081 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     4141 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     4201 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     4261 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     4321 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     4381 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     4441 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     4501 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     4561 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     4621 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     4681 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     4741 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     4801 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     4861 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     4921 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     4981 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     5041 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     5101 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     5161 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca
     5221 caaagaaggc tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat
     5281 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     5341 cgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     5401 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     5461 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     5521 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     5581 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     5641 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     5701 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     5761 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     5821 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     5881 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     5941 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     6001 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     6061 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     6121 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     6181 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     6241 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     6301 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     6361 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     6421 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     6481 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     6541 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     6601 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     6661 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     6721 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     6781 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     6841 ctctagcagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     6901 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     6961 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     7021 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     7081 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     7141 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     7201 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     7261 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     7321 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     7381 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     7441 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     7501 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     7561 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     7621 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     7681 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     7741 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     7801 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     7861 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     7921 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     7981 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
     8041 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     8101 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     8161 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     8221 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     8281 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     8341 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     8401 caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     8461 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     8521 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     8581 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     8641 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     8701 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     8761 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     8821 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     8881 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     8941 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     9001 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     9061 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     9121 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     9181 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     9241 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     9301 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     9361 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     9421 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     9481 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     9541 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     9601 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     9661 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     9721 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     9781 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     9841 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     9901 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     9961 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
    10021 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
    10081 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
    10141 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
    10201 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
    10261 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
    10321 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
    10381 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
    10441 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
    10501 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
    10561 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
    10621 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
    10681 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
    10741 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
    10801 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
    10861 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
    10921 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
    10981 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
    11041 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
    11101 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
    11161 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
    11221 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
    11281 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
    11341 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
    11401 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
    11461 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
    11521 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
    11581 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
    11641 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
    11701 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
    11761 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
    11821 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
    11881 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
    11941 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
    12001 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
    12061 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
    12121 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
    12181 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
    12241 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
    12301 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
    12361 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
    12421 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
    12481 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
    12541 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
    12601 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
    12661 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
    12721 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
    12781 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
    12841 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
    12901 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
    12961 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttg
//
