LOCUS       sequence-244551-       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_3
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    329..446
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        497..745
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     497..517
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     668..687
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        767..842
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        904..1115
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1134..1143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1140..5243
                     /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             1140..5243
                     /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             5244..5291
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5292..5315
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5325..5381
                     /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             5382..5978
                     /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     5875..5895
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5994..6582
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6047..6067)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6465..6476)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6654..6887
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6913..6930)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6919..7143
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7189..7617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7276..7295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7486..7507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7626..7646)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7631..7960
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7811..7946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7873..7892
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        8008..8055
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8074..8448
                     /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    8578..8699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8615..8634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8669..8688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8748..8764)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8748..8764)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8761..8783)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8772..8788
                     /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(8796..8802,8803..8820,8821..8826))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8841..8862
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8979..8996)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9150..9738)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9230..9249)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9909..10700,10701..10769))
                     /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     10532..10551
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10770..10874)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10949..10968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11140..11519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11521..11719
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11674..11694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11737..11917
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11964..12089
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12582..12815
                     /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(13000..13013,1..31)
                     /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 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
       61 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
      121 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
      181 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
      241 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
      301 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
      361 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
      421 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
      481 ggttaattaa ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
      541 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
      601 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
      661 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
      721 tatcttgtgg aaaggacgaa acaccgcgcc gcccgtgtac atccaggttt tagagctaga
      781 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      841 gcttttttga attcgctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
      901 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
      961 gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     1021 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     1081 ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca
     1141 tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga
     1201 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     1261 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     1321 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     1381 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     1441 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     1501 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     1561 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     1621 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     1681 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     1741 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     1801 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     1861 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     1921 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     1981 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     2041 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     2101 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     2161 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     2221 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     2281 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     2341 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     2401 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     2461 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     2521 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     2581 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     2641 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     2701 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     2761 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     2821 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     2881 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     2941 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     3001 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     3061 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     3121 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     3181 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     3241 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     3301 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     3361 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     3421 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     3481 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     3541 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     3601 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     3661 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     3721 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     3781 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     3841 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     3901 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     3961 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     4021 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     4081 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     4141 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     4201 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     4261 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     4321 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     4381 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     4441 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     4501 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     4561 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     4621 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     4681 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     4741 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     4801 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     4861 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     4921 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     4981 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     5041 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     5101 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     5161 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     5221 acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta
     5281 agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc
     5341 tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg
     5401 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     5461 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     5521 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     5581 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     5641 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     5701 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     5761 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     5821 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     5881 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     5941 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac
     6001 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     6061 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     6121 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     6181 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     6241 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     6301 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     6361 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     6421 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     6481 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     6541 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat
     6601 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     6661 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     6721 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6781 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6841 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa
     6901 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     6961 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     7021 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     7081 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     7141 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     7201 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     7261 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     7321 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     7381 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     7441 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     7501 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     7561 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     7621 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     7681 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     7741 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     7801 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     7861 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     7921 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     7981 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     8041 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
     8101 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
     8161 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
     8221 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
     8281 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
     8341 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
     8401 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
     8461 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     8521 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     8581 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     8641 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     8701 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     8761 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     8821 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     8881 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     8941 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     9001 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     9061 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     9121 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     9181 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     9241 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     9301 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     9361 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     9421 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     9481 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     9541 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     9601 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     9661 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     9721 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     9781 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     9841 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     9901 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     9961 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    10021 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    10081 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
    10141 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
    10201 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
    10261 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
    10321 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
    10381 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    10441 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    10501 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    10561 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    10621 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    10681 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    10741 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    10801 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    10861 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
    10921 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
    10981 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
    11041 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
    11101 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
    11161 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
    11221 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
    11281 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
    11341 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
    11401 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
    11461 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
    11521 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
    11581 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
    11641 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
    11701 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
    11761 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
    11821 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
    11881 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
    11941 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
    12001 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
    12061 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
    12121 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
    12181 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
    12241 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
    12301 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
    12361 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
    12421 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
    12481 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
    12541 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
    12601 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
    12661 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
    12721 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
    12781 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
    12841 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
    12901 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
    12961 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca aga
//
