LOCUS       sequence-244544-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting human 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_1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    247..364
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        415..663
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     415..435
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     586..605
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        685..760
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        822..1033
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1052..1061
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1058..5161
                     /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             1058..5161
                     /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             5162..5209
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5210..5233
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5243..5299
                     /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             5300..5896
                     /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     5793..5813
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5912..6500
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5965..5985)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6383..6394)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6572..6805
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6831..6848)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6837..7061
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7107..7535
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7194..7213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7404..7425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7544..7564)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7549..7878
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7729..7864
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7791..7810
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7926..7973
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7992..8366
                     /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    8496..8617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8533..8552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8587..8606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8666..8682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8666..8682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8679..8701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8690..8706
                     /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(8714..8720,8721..8738,8739..8744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8759..8780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8897..8914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9068..9656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9148..9167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9827..10618,10619..10687))
                     /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     10450..10469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10688..10792)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10867..10886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11058..11437
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11439..11637
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11592..11612
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11655..11835
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11882..12007
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12500..12733
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12918..12962
                     /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 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
       61 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
      121 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt
      181 cgattagtga acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat
      241 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
      301 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
      361 ttttcgggtt tattacaggg acagcagaga tccagtttgg ttaattaagg taccgagggc
      421 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      481 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      541 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      601 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
      661 accgggtgtg gtcgtagcct gcaggtttta gagctagaaa tagcaagtta aaataaggct
      721 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgaat tcgctagcta
      781 ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc
      841 cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg
      901 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
      961 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     1021 cgccagaaca caggaccggt tctagagcgc tgccaccatg gacaagaagt acagcatcgg
     1081 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     1141 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     1201 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1261 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     1321 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     1381 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     1441 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     1501 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     1561 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     1621 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     1681 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     1741 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     1801 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     1861 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     1921 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     1981 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     2041 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     2101 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     2161 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     2221 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     2281 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     2341 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     2401 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     2461 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     2521 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     2581 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     2641 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     2701 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     2761 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     2821 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     2881 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     2941 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     3001 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     3061 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     3121 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     3181 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     3241 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     3301 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     3361 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     3421 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     3481 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     3541 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     3601 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     3661 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     3721 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     3781 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     3841 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     3901 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     3961 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     4021 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     4081 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     4141 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     4201 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     4261 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     4321 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     4381 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     4441 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     4501 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     4561 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     4621 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     4681 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     4741 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     4801 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     4861 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     4921 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     4981 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     5041 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     5101 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     5161 caagcgacct gccgccacaa agaaggctgg acaggctaag aagaagaaag attacaaaga
     5221 cgatgacgat aagggatccg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt
     5281 cgaagagaat cctggaccga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     5341 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     5401 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     5461 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     5521 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     5581 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     5641 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca
     5701 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     5761 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     5821 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     5881 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     5941 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     6001 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     6061 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     6121 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     6181 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     6241 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     6301 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     6361 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     6421 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     6481 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     6541 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     6601 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     6661 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     6721 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     6781 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
     6841 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     6901 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     6961 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     7021 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     7081 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     7141 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     7201 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     7261 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     7321 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     7381 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     7441 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     7501 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     7561 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     7621 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     7681 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     7741 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     7801 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     7861 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
     7921 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     7981 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     8041 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     8101 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     8161 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     8221 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     8281 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     8341 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
     8401 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     8461 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     8521 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     8581 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     8641 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     8701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     8761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     8821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     8881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     8941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     9001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     9061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     9121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     9181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     9241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     9301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     9361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     9421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     9481 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     9541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     9601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     9661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     9721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     9781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     9841 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     9901 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     9961 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
    10021 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
    10081 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
    10141 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
    10201 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
    10261 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
    10321 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
    10381 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
    10441 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
    10501 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
    10561 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
    10621 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
    10681 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
    10741 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
    10801 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
    10861 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
    10921 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
    10981 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
    11041 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
    11101 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
    11161 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
    11221 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
    11281 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
    11341 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
    11401 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
    11461 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
    11521 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
    11581 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagcgc
    11641 gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
    11701 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
    11761 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
    11821 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
    11881 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
    11941 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
    12001 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
    12061 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
    12121 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
    12181 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
    12241 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
    12301 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
    12361 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
    12421 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
    12481 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
    12541 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
    12601 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
    12661 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
    12721 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
    12781 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
    12841 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
    12901 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
    12961 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ata
//
