LOCUS       sequence-244569-       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_1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    319..436
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        487..735
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     487..507
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     658..677
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        757..832
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        894..1105
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1124..1133
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1130..5233
                     /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             1130..5233
                     /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             5234..5281
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5282..5305
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5315..5371
                     /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             5372..5968
                     /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     5865..5885
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5984..6572
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6037..6057)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6455..6466)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6644..6877
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6903..6920)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6909..7133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7179..7607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7266..7285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7476..7497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7616..7636)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7621..7950
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7801..7936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7863..7882
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7998..8045
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8064..8438
                     /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    8568..8689
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8605..8624)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8659..8678
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8738..8754)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8738..8754)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8751..8773)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8762..8778
                     /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(8786..8792,8793..8810,8811..8816))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8831..8852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8969..8986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9140..9728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9220..9239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9899..10690,10691..10759))
                     /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     10522..10541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10760..10864)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10939..10958)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11130..11509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11511..11709
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11664..11684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11727..11907
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11954..12079
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12572..12805
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             join(12990..13013,1..21)
                     /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 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
       61 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
      121 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
      181 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
      241 gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat
      301 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
      361 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
      421 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttaattaa
      481 ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      541 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      601 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      661 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      721 aaaggacgaa acaccgtgga gttgagatca tacatggttt tagagctaga aatagcaagt
      781 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttga
      841 attcgctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga
      901 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc
      961 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     1021 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1081 caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca tggacaagaa
     1141 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1201 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1261 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1321 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1381 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1441 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1501 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1561 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1621 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1681 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1741 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1801 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     1861 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     1921 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     1981 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     2041 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2101 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2161 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2221 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2281 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2341 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2401 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2461 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2521 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2581 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2641 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2701 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     2761 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     2821 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     2881 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     2941 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     3001 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3061 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3121 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3181 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3241 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3301 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3361 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3421 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3481 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3541 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3601 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3661 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3721 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3781 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     3841 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     3901 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     3961 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     4021 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4081 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4141 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4201 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4261 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4321 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4381 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4441 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4501 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4561 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4621 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4681 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4741 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4801 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     4861 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     4921 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     4981 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     5041 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5101 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5161 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5221 gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa
     5281 agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc tgctgaaaca
     5341 agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg tgcgcctcgc
     5401 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     5461 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     5521 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     5581 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     5641 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     5701 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     5761 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     5821 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     5881 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     5941 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
     6001 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     6061 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     6121 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     6181 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     6241 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     6301 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     6361 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     6421 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     6481 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     6541 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     6601 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     6661 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     6721 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     6781 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     6841 ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc
     6901 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6961 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     7021 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     7081 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     7141 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     7201 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     7261 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     7321 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     7381 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     7441 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     7501 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     7561 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     7621 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     7681 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     7741 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     7801 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     7861 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     7921 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     7981 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     8041 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
     8101 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     8161 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     8221 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     8281 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     8341 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     8401 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
     8461 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     8521 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     8581 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     8641 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     8701 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     8761 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     8821 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     8881 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     8941 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     9001 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     9061 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     9121 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     9181 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     9241 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     9301 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     9361 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     9421 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     9481 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     9541 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     9601 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     9661 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     9721 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     9781 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     9841 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     9901 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     9961 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    10021 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    10081 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    10141 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    10201 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    10261 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    10321 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    10381 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    10441 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    10501 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    10561 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    10621 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    10681 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    10741 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    10801 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    10861 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
    10921 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
    10981 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
    11041 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
    11101 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
    11161 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
    11221 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
    11281 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
    11341 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
    11401 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
    11461 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
    11521 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
    11581 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
    11641 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
    11701 tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
    11761 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
    11821 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
    11881 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
    11941 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
    12001 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
    12061 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
    12121 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
    12181 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
    12241 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
    12301 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
    12361 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
    12421 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
    12481 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
    12541 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
    12601 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
    12661 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
    12721 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
    12781 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
    12841 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
    12901 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
    12961 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaa
//
