LOCUS       sequence-244548-       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_2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    305..422
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        473..721
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     473..493
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     644..663
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        743..818
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        880..1091
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1110..1119
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1116..5219
                     /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             1116..5219
                     /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             5220..5267
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5268..5291
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5301..5357
                     /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             5358..5954
                     /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     5851..5871
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5970..6558
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6023..6043)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6441..6452)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6630..6863
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6889..6906)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6895..7119
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7165..7593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7252..7271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7462..7483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7602..7622)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7607..7936
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7787..7922
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7849..7868
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7984..8031
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8050..8424
                     /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    8554..8675
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8591..8610)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8645..8664
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8724..8740)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8724..8740)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8737..8759)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8748..8764
                     /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(8772..8778,8779..8796,8797..8802))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8817..8838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8955..8972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9126..9714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9206..9225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9885..10676,10677..10745))
                     /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     10508..10527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10746..10850)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10925..10944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11116..11495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11497..11695
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11650..11670
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11713..11893
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11940..12065
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12558..12791
                     /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(12976..13013,1..7)
                     /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 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
       61 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
      121 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
      181 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
      241 attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc
      301 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
      361 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
      421 ttcgggttta ttacagggac agcagagatc cagtttggtt aattaaggta ccgagggcct
      481 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
      541 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      601 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      661 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      721 cgactgccca ccgtgcctgg gggttttaga gctagaaata gcaagttaaa ataaggctag
      781 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc gctagctagg
      841 tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      901 cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc
      961 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     1021 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     1081 ccagaacaca ggaccggttc tagagcgctg ccaccatgga caagaagtac agcatcggcc
     1141 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     1201 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     1261 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
     1321 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     1381 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     1441 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     1501 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     1561 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     1621 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     1681 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     1741 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     1801 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga
     1861 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     1921 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     1981 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     2041 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     2101 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     2161 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
     2221 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     2281 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     2341 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     2401 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     2461 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     2521 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
     2581 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     2641 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
     2701 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg
     2761 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     2821 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     2881 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     2941 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     3001 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     3061 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     3121 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     3181 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     3241 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     3301 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     3361 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     3421 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     3481 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     3541 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     3601 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg
     3661 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca
     3721 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg
     3781 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     3841 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     3901 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc
     3961 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     4021 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
     4081 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
     4141 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
     4201 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
     4261 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
     4321 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca
     4381 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
     4441 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta
     4501 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
     4561 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
     4621 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
     4681 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
     4741 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
     4801 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
     4861 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga
     4921 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
     4981 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata
     5041 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
     5101 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
     5161 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca
     5221 agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagat tacaaagacg
     5281 atgacgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg
     5341 aagagaatcc tggaccgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     5401 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     5461 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     5521 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     5581 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     5641 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     5701 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
     5761 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     5821 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     5881 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     5941 agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     6001 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     6061 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     6121 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     6181 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     6241 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     6301 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     6361 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     6421 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     6481 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     6541 tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct
     6601 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     6661 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6721 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6781 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6841 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     6901 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     6961 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     7021 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     7081 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     7141 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     7201 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     7261 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     7321 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     7381 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     7441 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     7501 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     7561 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     7621 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     7681 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     7741 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     7801 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     7861 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     7921 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     7981 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     8041 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc
     8101 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac
     8161 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
     8221 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag
     8281 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc
     8341 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac
     8401 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc
     8461 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     8521 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     8581 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     8641 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     8701 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     8761 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     8821 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     8881 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     8941 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     9001 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     9061 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     9121 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     9181 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     9241 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     9301 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     9361 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     9421 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     9481 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     9541 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     9601 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     9661 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     9721 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     9781 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     9841 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     9901 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     9961 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
    10021 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
    10081 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
    10141 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
    10201 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
    10261 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
    10321 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
    10381 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
    10441 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
    10501 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
    10561 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
    10621 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
    10681 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
    10741 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
    10801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
    10861 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
    10921 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
    10981 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
    11041 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
    11101 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
    11161 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
    11221 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
    11281 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
    11341 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
    11401 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
    11461 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
    11521 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
    11581 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
    11641 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt
    11701 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
    11761 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
    11821 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
    11881 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
    11941 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
    12001 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
    12061 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
    12121 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
    12181 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
    12241 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
    12301 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
    12361 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
    12421 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
    12481 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
    12541 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
    12601 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
    12661 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
    12721 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
    12781 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
    12841 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
    12901 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
    12961 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggc
//
