LOCUS       sequence-244568-       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_4
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    279..396
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        447..695
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     447..467
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     618..637
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        717..792
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        854..1065
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1084..1093
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1090..5193
                     /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             1090..5193
                     /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             5194..5241
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5242..5265
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5275..5331
                     /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             5332..5928
                     /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     5825..5845
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5944..6532
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5997..6017)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6415..6426)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6604..6837
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6863..6880)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6869..7093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7139..7567
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7226..7245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7436..7457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7576..7596)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7581..7910
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7761..7896
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7823..7842
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7958..8005
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8024..8398
                     /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    8528..8649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8565..8584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8619..8638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8698..8714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8698..8714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8711..8733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8722..8738
                     /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(8746..8752,8753..8770,8771..8776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8791..8812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8929..8946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9100..9688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9180..9199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9859..10650,10651..10719))
                     /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     10482..10501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10720..10824)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10899..10918)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11090..11469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11471..11669
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11624..11644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11687..11867
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11914..12039
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12532..12765
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12950..12994
                     /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 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
       61 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
      121 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
      181 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg
      241 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
      301 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
      361 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
      421 gatccagttt ggttaattaa ggtaccgagg gcctatttcc catgattcct tcatatttgc
      481 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
      541 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
      601 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
      661 tggctttata tatcttgtgg aaaggacgaa acaccggcta tgtctggaat ctgacggttt
      721 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      781 ccgagtcggt gcttttttga attcgctagc taggtcttga aaggagtggg aattggctcc
      841 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      901 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      961 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     1021 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagagc
     1081 gctgccacca tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     1141 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     1201 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     1261 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     1321 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     1381 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     1441 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     1501 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     1561 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     1621 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     1681 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     1741 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     1801 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     1861 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     1921 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     1981 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     2041 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     2101 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     2161 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     2221 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     2281 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     2341 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     2401 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     2461 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     2521 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     2581 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     2641 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     2701 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     2761 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     2821 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     2881 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     2941 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     3001 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     3061 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     3121 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     3181 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     3241 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     3301 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     3361 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     3421 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     3481 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     3541 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     3601 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     3661 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     3721 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     3781 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     3841 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     3901 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     3961 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     4021 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     4081 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     4141 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     4201 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     4261 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     4321 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     4381 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     4441 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     4501 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     4561 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     4621 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     4681 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     4741 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     4801 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     4861 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     4921 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     4981 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     5041 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     5101 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     5161 acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct
     5221 ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca
     5281 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac
     5341 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     5401 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     5461 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     5521 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     5581 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     5641 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     5701 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     5761 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     5821 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     5881 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     5941 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     6001 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     6061 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     6121 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     6181 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     6241 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     6301 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     6361 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     6421 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     6481 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     6541 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     6601 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     6661 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     6721 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     6781 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
     6841 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     6901 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6961 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     7021 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     7081 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     7141 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     7201 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     7261 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     7321 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     7381 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     7441 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     7501 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     7561 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     7621 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     7681 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     7741 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     7801 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     7861 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     7921 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
     7981 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
     8041 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     8101 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     8161 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     8221 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     8281 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     8341 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
     8401 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     8461 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     8521 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     8581 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     8641 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     8701 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     8761 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     8821 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     8881 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     8941 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     9001 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     9061 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     9121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     9181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     9241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     9301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     9361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     9421 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     9481 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     9541 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     9601 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     9661 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     9721 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     9781 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     9841 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     9901 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     9961 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    10021 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    10081 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    10141 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    10201 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    10261 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    10321 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    10381 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    10441 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    10501 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    10561 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    10621 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    10681 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    10741 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    10801 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
    10861 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
    10921 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
    10981 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
    11041 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
    11101 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
    11161 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
    11221 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
    11281 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
    11341 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
    11401 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
    11461 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
    11521 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
    11581 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
    11641 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
    11701 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
    11761 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
    11821 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
    11881 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
    11941 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
    12001 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
    12061 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
    12121 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
    12181 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
    12241 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
    12301 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
    12361 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
    12421 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
    12481 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
    12541 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
    12601 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
    12661 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
    12721 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
    12781 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
    12841 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
    12901 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
    12961 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taa
//
