LOCUS       sequence-260836-       13128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of Cas9 and gRNA targeting PBRM1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13128)
  AUTHORS   Gao W, Li W, Xiao T, Liu XS, Kaelin WG Jr
  TITLE     Inactivation of the PBRM1 tumor suppressor gene amplifies the 
            HIF-response in VHL-/- clear cell renal carcinoma.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):1027-1032. doi: 
            10.1073/pnas.1619726114. Epub 2017 Jan 12.
  PUBMED    28082722
REFERENCE   2  (bases 1 to 13128)
  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.1619726114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-01-31- 31"; volume: "114"; issue: "5"; pages: 
            "1027-1032"
COMMENT     Sequence Label: sgPBRM1-1- LentiCRISPRv2
FEATURES             Location/Qualifiers
     source          1..13128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..95)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        80..409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      260..395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     322..341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        457..504
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             523..897
                     /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    1027..1148
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1064..1083)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1118..1137
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1197..1213)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1197..1213)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1210..1232)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1221..1237
                     /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(1245..1251,1252..1269,1270..1275))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1290..1311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1428..1445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1599..2187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1679..1698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2358..3149,3150..3218))
                     /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     2981..3000
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3219..3323)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3398..3417)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3589..3968
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3970..4168
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4123..4143
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4186..4366
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4413..4538
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5031..5264
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5449..5493
                     /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    5791..5908
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    5974..5998
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     6161..6180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6258..6333
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(6379..6403)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        6481..6692
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6711..6720
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6717..10820
                     /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             6717..10820
                     /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             10821..10868
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10869..10892
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10902..10958
                     /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             10959..11555
                     /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     11452..11472
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11571..12159
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11624..11644)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12042..12053)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12231..12464
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12490..12507)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12496..12720
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(12766..13128,1..66)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12853..12872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13063..13084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
       61 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
      121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
      181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
      241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
      301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
      361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
      421 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
      481 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
      541 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
      601 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
      661 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
      721 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
      781 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
      841 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac
      901 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
      961 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     1021 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1081 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1141 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     1201 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1261 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     1321 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1381 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1441 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1501 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1561 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1621 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1681 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1741 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1801 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1861 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1921 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1981 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2041 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2101 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2161 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2221 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2281 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2341 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2401 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2461 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2521 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2581 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2641 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2701 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2761 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2821 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2881 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2941 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3001 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3061 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3121 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3181 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3241 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3301 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     3361 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     3421 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     3481 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     3541 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     3601 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     3661 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     3721 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     3781 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     3841 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     3901 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     3961 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     4021 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     4081 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     4141 gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta
     4201 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     4261 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     4321 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     4381 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     4441 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     4501 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     4561 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     4621 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     4681 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     4741 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     4801 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     4861 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     4921 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     4981 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     5041 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     5101 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     5161 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     5221 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     5281 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     5341 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     5401 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     5461 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     5521 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     5581 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     5641 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     5701 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     5761 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     5821 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     5881 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     5941 ttggttaatt aacccacaag gagacgacct tccacaagtt tgtacaaaaa agcaggcttt
     6001 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     6061 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     6121 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     6181 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc
     6241 gaggagatct atatcttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     6301 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattctagat cttgagacaa
     6361 atggcagtat tcatccacac ccagctttct tgtacaaagt ggtgggtgag gcggttcagg
     6421 tggcggagga agctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
     6481 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
     6541 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     6601 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     6661 tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct gccaccatgg
     6721 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     6781 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     6841 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     6901 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     6961 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     7021 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     7081 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     7141 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     7201 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     7261 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     7321 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     7381 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     7441 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     7501 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     7561 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     7621 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     7681 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     7741 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     7801 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     7861 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     7921 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     7981 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     8041 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     8101 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     8161 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     8221 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     8281 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     8341 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     8401 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     8461 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     8521 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     8581 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     8641 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     8701 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     8761 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     8821 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     8881 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     8941 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     9001 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     9061 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     9121 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     9181 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
     9241 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
     9301 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     9361 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
     9421 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     9481 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     9541 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     9601 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     9661 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     9721 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     9781 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     9841 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     9901 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     9961 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
    10021 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
    10081 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
    10141 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
    10201 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
    10261 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
    10321 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
    10381 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
    10441 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
    10501 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
    10561 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
    10621 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
    10681 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
    10741 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
    10801 tgtctcagct gggaggcgac aagcgacctg ccgccacaaa gaaggctgga caggctaaga
    10861 agaagaaaga ttacaaagac gatgacgata agggatccgg cgcaacaaac ttctctctgc
    10921 tgaaacaagc cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc
    10981 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
    11041 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
    11101 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
    11161 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
    11221 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
    11281 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
    11341 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
    11401 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
    11461 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
    11521 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
    11581 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
    11641 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
    11701 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
    11761 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
    11821 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
    11881 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
    11941 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
    12001 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
    12061 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
    12121 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
    12181 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
    12241 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
    12301 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
    12361 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
    12421 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
    12481 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
    12541 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
    12601 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
    12661 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
    12721 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
    12781 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
    12841 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
    12901 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
    12961 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
    13021 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
    13081 ctggaacaac actcaaccct atctcggtct attcttttga tttataag
//
