LOCUS       sequence-180819-       11589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNAs targeting mouse Kras. 3rd generation lentiviral backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11589)
  AUTHORS   Mou H, Moore J, Malonia SK, Li Y, Ozata DM, Hough S, Song CQ, Smith 
            JL, Fischer A, Weng Z, Green MR, Xue W
  TITLE     Genetic disruption of oncogenic Kras sensitizes lung cancer cells to
            Fas receptor-mediated apoptosis.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Apr 4;114(14):3648-3653. doi: 
            10.1073/pnas.1620861114. Epub 2017 Mar 20.
  PUBMED    28320962
REFERENCE   2  (bases 1 to 11589)
  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: 
            "10.1073/pnas.1620861114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-04-4- 4"; volume: "114"; issue: "14"; pages: "3648-3653"
COMMENT     Sequence Label: lentiCRISPR.sgKras.9
FEATURES             Location/Qualifiers
     source          1..11589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    393..510
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        579..790
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      810..819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             819..842
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             849..869
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             894..4994
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             894..4994
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4995..5042
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5058..5114
                     /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             5115..5714
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5115..5134)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5611..5631
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5730..6318
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5783..5803)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6201..6212)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6390..6623
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6701..6822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6738..6757)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6792..6811
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6862..6997
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7017..7036)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7018..7036)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7046..7063)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7046..7062)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7055..7077)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7204..7659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7291..7310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7501..7522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7685..7789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7790..7858,7859..8650)
                     /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     complement(8008..8027)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8821..9409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9310..9329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9563..9580
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9697..9718
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9733..9738,9739..9756,9757..9763)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9771..9787
                     /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)."
     primer_bind     9776..9798
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9795..9811
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9795..9811
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9830..9850
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9832..9850
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9878..10104
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10105..10285
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10332..10457
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10950..11183
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11368..11412
                     /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 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tggttggagc tgatggcgtg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattctag
      361 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
      421 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
      481 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
      541 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
      601 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
      661 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
      721 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
      781 cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata
      841 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
      901 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
      961 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1021 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1081 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1141 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1201 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1261 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1321 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1381 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1441 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1501 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1561 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     1621 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     1681 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     1741 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     1801 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     1861 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     1921 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     1981 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2041 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2101 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2161 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2221 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2281 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2341 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2401 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2461 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2521 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     2581 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     2641 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     2701 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     2761 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     2821 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     2881 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     2941 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3001 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3061 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3121 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3181 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3241 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3301 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3361 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3421 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3481 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3541 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     3601 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     3661 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     3721 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     3781 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     3841 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     3901 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     3961 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4021 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4081 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4141 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4201 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4261 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4321 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4381 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4441 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4501 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4561 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     4621 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     4681 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     4741 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     4801 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     4861 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     4921 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     4981 agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga
     5041 aagctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc gacgtggagg
     5101 agaaccccgg ccccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     5161 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     5221 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     5281 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     5341 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     5401 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     5461 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
     5521 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     5581 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     5641 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     5701 agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     5761 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     5821 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     5881 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     5941 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     6001 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     6061 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     6121 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     6181 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     6241 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     6301 tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct
     6361 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     6421 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6481 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6541 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6601 gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta
     6661 tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta
     6721 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     6781 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct
     6841 atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     6901 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     6961 ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct
     7021 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     7081 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     7141 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     7201 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7261 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7381 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     7441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7501 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     7561 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     7621 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     7681 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     7741 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     7801 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     7861 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     7921 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     7981 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     8041 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     8101 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     8161 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     8221 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     8281 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     8341 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     8401 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     8461 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     8521 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     8581 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     8641 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     8701 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     8761 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     8821 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     8881 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     8941 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     9001 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     9061 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     9121 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     9181 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     9241 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     9301 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     9361 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     9421 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     9481 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     9541 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     9601 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     9661 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     9721 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     9781 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     9841 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     9901 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     9961 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
    10021 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
    10081 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
    10141 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
    10201 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
    10261 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
    10321 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
    10381 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
    10441 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
    10501 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
    10561 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
    10621 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
    10681 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
    10741 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
    10801 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
    10861 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
    10921 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
    10981 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
    11041 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
    11101 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
    11161 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
    11221 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
    11281 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
    11341 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
    11401 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
    11461 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
    11521 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
    11581 ggacccaga
//
