LOCUS       sequence-238737-       13028 bp    DNA     circular SYN 15-APR-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 13028)
  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 13028)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..13028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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"
     misc_feature    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2005..2253
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2005..2025
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2176..2195
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2275..2350
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2412..2623
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2642..2651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2648..6751
                     /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             2648..6751
                     /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             6752..6799
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6800..6823
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6833..6889
                     /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             6890..7486
                     /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     7383..7403
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7502..8090
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7555..7575)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7973..7984)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8162..8395
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8421..8438)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8427..8651
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8697..9125
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8784..8803)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8994..9015
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9134..9154)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9139..9468
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9319..9454
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9381..9400
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9516..9563
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9582..9956
                     /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    10086..10207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10123..10142)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10177..10196
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10256..10272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10256..10272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10269..10291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10280..10296
                     /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(10304..10310,10311..10328,10329..10334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10349..10370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10487..10504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10658..11246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10738..10757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11417..12208,12209..12277))
                     /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     12040..12059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12278..12382)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12457..12476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12648..13027
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtacca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
     2041 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2101 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2161 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2221 gctttatata tcttgtggaa aggacgaaac accgactgcc caccgtgcct gggggtttta
     2281 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     2341 gagtcggtgc ttttttgaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg
     2401 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     2461 cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     2521 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     2581 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc
     2641 tgccaccatg gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     2701 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     2761 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     2821 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     2881 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     2941 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     3001 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     3061 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     3121 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     3181 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     3241 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     3301 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     3361 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     3421 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     3481 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     3541 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     3601 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     3661 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     3721 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     3781 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     3841 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     3901 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     3961 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     4021 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     4081 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
     4141 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     4201 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     4261 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     4321 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     4381 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     4441 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     4501 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     4561 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     4621 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     4681 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     4741 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     4801 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     4861 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     4921 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     4981 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     5041 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     5101 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     5161 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
     5221 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     5281 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     5341 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     5401 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     5461 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     5521 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     5581 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     5641 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     5701 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     5761 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     5821 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     5881 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     5941 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     6001 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     6061 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     6121 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     6181 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     6241 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     6301 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     6361 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     6421 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     6481 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     6541 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     6601 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     6661 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     6721 acggatcgac ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg
     6781 acaggctaag aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa
     6841 cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccga ccgagtacaa
     6901 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     6961 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     7021 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     7081 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     7141 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     7201 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     7261 ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     7321 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     7381 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     7441 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga
     7501 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7561 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7621 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7681 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7741 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7801 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     7861 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     7921 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     7981 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     8041 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     8101 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     8161 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     8221 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8281 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8341 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
     8401 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     8461 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     8521 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     8581 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     8641 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     8701 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     8761 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     8821 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     8881 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     8941 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     9001 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     9061 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     9121 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     9181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     9241 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     9301 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     9361 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     9421 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     9481 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
     9541 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
     9601 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     9661 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     9721 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     9781 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     9841 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     9901 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     9961 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
    10021 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
    10081 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
    10141 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
    10201 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
    10261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
    10321 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
    10381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
    10441 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
    10501 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
    10561 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
    10621 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    10681 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    10741 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    10801 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    10861 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    10921 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    10981 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    11041 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    11101 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    11161 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    11221 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    11281 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    11341 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    11401 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    11461 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    11521 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    11581 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    11641 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    11701 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    11761 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    11821 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    11881 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    11941 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    12001 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    12061 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    12121 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    12181 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    12241 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    12301 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    12361 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
    12421 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
    12481 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
    12541 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
    12601 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
    12661 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
    12721 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
    12781 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
    12841 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
    12901 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
    12961 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
    13021 taccatgg
//
