LOCUS       sequence-273705-       12047 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses a mouse LKB1-targeting sgRNA, Cas9, and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12047)
  AUTHORS   Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera 
            LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE, 
            Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ
  TITLE     The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive
            Effects of LKB1 in NSCLC.
  JOURNAL   Cancer Discov. 2019 Nov;9(11):1606-1627. doi: 
            10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26.
  PUBMED    31350328
REFERENCE   2  (bases 1 to 12047)
  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: 
            "10.1158/2159-8290.CD-18-1261"; journalName: "Cancer Discov"; date: 
            "2019-11"; volume: "9"; issue: "11"; pages: "1606-1627"
COMMENT     Sequence Label: pSECC-sgLKB1-A
FEATURES             Location/Qualifiers
     source          1..12047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /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    2443..2560
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2660..2908
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2660..2680
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2831..2850
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2930..3005
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3053..3170
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3269..3480
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3525..3534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3534..3557
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3564..3584
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3609..7709
                     /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             3609..7709
                     /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             7710..7757
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7773..7829
                     /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             7833..7853
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7854..8882
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPGIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    8935..9523
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8988..9008)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9406..9417)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9526..9542)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9527..9543)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10052..10232
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(10294..10882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10374..10393)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11053..11844,11845..11913))
                     /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     11676..11695
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11914..12018)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2581 cagagatcca gtttggttag taccgggccc gggtcgacta gtgatcagtg tgagggagtg
     2641 taaagctggg tttcccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2701 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     2761 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2821 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2881 atatatcttg tggaaaggac gaaacaccgc gtacgagccc tccccgagcg ttttagagct
     2941 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     3001 ggtgcttttt tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa
     3061 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     3121 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     3181 acagggacag cagagatcca ctttggcgcc ggcaaaccgc tgttcctagg aatcccgagg
     3241 ccttcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     3301 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     3361 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     3421 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3481 gtgtcgtgac gcgggacccg acattagcgc tacagcttaa gcgggccacc atggattaca
     3541 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     3601 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     3661 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     3721 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     3781 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     3841 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     3901 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     3961 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     4021 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     4081 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     4141 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     4201 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     4261 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     4321 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     4381 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     4441 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     4501 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     4561 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     4621 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     4681 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     4741 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     4801 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     4861 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     4921 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     4981 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     5041 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     5101 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     5161 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     5221 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     5281 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     5341 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     5401 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     5461 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     5521 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     5581 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     5641 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     5701 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     5761 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     5821 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     5881 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     5941 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     6001 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     6061 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     6121 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     6181 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     6241 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     6301 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     6361 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     6421 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     6481 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     6541 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     6601 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     6661 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     6721 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     6781 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     6841 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     6901 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     6961 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     7021 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     7081 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     7141 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     7201 ctaagtactc cctgttcgag ctggaaaacg gcaggaagag aatgctggcc tctgccggcg
     7261 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     7321 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     7381 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     7441 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     7501 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     7561 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     7621 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     7681 ggatcgacct gtctcagctg ggaggcgaca agcgtcctgc tgctactaag aaagctggtc
     7741 aagctaagaa aaagaaagct agcggcagcg gcgccaccaa cttcagcctg ctgaagcagg
     7801 ccggcgacgt ggaggagaac cccggcccca tgcccaagaa gaagaggaag gtgtccaatt
     7861 tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc
     7921 gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa
     7981 tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt
     8041 ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg
     8101 cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc
     8161 tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa
     8221 acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc
     8281 aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc
     8341 tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag
     8401 atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg
     8461 ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat
     8521 ggatttccgt atctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa
     8581 aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga
     8641 tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc
     8701 tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag
     8761 tttcaatacc ggggatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact
     8821 atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatt
     8881 agaaacgttg ttgtttgggg ttgaattact cttctagacc gcggcgcgcc cgataatcaa
     8941 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     9001 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     9061 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     9121 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     9181 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     9241 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     9301 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     9361 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     9421 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     9481 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct
     9541 cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
     9601 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
     9661 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     9721 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
     9781 gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat
     9841 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt
     9901 agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat
     9961 ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca
    10021 catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc
    10081 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
    10141 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
    10201 acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc
    10261 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
    10321 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
    10381 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
    10441 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
    10501 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
    10561 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
    10621 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
    10681 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
    10741 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
    10801 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
    10861 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
    10921 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
    10981 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
    11041 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    11101 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    11161 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    11221 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    11281 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    11341 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    11401 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    11461 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    11521 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    11581 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    11641 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    11701 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    11761 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    11821 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    11881 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    11941 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    12001 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacg
//
