LOCUS       sequence-464638-       13212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knock out of murine AGAP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13212)
  AUTHORS   Nikolatou K, Sandilands E, Roman-Fernandez A, Cumming EM, Freckmann 
            E, Lilla S, Buetow L, McGarry L, Neilson M, Shaw R, Strachan D, 
            Miller C, Huang DT, McNeish IA, Norman JC, Zanivan S, Bryant DM
  TITLE     PTEN deficiency exposes a requirement for an ARF GTPase module for 
            integrin-dependent invasion in ovarian cancer.
  JOURNAL   EMBO J. 2023 Sep 18;42(18):e113987. doi: 10.15252/embj.2023113987. 
            Epub 2023 Aug 14.
  PUBMED    37577760
REFERENCE   2  (bases 1 to 13212)
  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"; journalName: "EMBO J."; 
            date: "2023-09-18"; volume: "42(18):e113987. doi"; pages: " 
            10.15252/embj.2023113987. Epub 2023 Aug 14"
COMMENT     Sequence Label: pLentiCRISPR V2 Neo sgAGAP1_3
FEATURES             Location/Qualifiers
     source          1..13212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        242..621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     776..796
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             839..1019
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1066..1191
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2612..2860
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2612..2632
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2783..2802
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2882..2957
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3019..3230
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3249..3258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3255..7358
                     /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             3255..7358
                     /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             7359..7406
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7407..7430
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7440..7496
                     /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             7497..8288
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7548..7567)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8158..8177
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    8304..8892
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8357..8377)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8775..8786)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8964..9197
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9223..9240)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9229..9453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9499..9927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9586..9605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9796..9817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9936..9956)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9941..10270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10121..10256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10183..10202
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10318..10365
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10384..10758
                     /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    10888..11009
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10925..10944)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10979..10998
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11058..11074)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11058..11074)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11071..11093)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11082..11098
                     /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(11106..11112,11113..11130,11131..11136))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11151..11172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11289..11306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11460..12048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11540..11559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12219..13010,13011..13079))
                     /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     12842..12861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13080..13184)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgcgcgt
      241 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      301 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      361 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      421 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      481 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      541 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      601 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      661 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      721 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg
      841 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      901 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      961 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     1021 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     1081 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     1141 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     1201 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1261 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1321 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1381 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1441 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1501 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata
     2641 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     2701 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2761 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2821 tttcttggct ttatatatct tgtggaaagg acgaaacacc gtacgtcacc ctgtgcgaca
     2881 agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     2941 tggcaccgag tcggtgcttt tttgaattcg ctagctaggt cttgaaagga gtgggaattg
     3001 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     3061 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     3121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     3181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggttct
     3241 agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg
     3301 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     3361 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     3421 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     3481 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     3541 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     3601 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     3661 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     3721 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     3781 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     3841 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     3901 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     3961 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     4021 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     4081 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     4141 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     4201 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     4261 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     4321 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     4381 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     4441 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     4501 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     4561 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     4621 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     4681 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     4741 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     4801 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     4861 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     4921 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     4981 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     5041 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     5101 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     5161 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     5221 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     5281 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     5341 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     5401 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     5461 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     5521 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     5581 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     5641 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     5701 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     5761 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     5821 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     5881 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     5941 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     6001 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     6061 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     6121 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     6181 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     6241 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     6301 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     6361 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     6421 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     6481 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     6541 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     6601 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     6661 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     6721 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     6781 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     6841 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     6901 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     6961 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     7021 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     7081 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     7141 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     7201 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     7261 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     7321 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     7381 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggcg
     7441 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgattg
     7501 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     7561 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     7621 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     7681 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     7741 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     7801 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     7861 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     7921 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     7981 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     8041 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     8101 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     8161 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     8221 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     8281 tcttctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     8341 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     8401 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     8461 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     8521 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     8581 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     8641 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     8701 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     8761 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     8821 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     8881 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     8941 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     9001 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     9061 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     9121 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     9181 gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     9241 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     9301 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     9361 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     9421 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     9481 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     9541 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     9601 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     9661 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     9721 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     9781 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     9841 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     9901 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     9961 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
    10021 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
    10081 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
    10141 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
    10201 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
    10261 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
    10321 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
    10381 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg
    10441 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc
    10501 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg
    10561 gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg
    10621 gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag
    10681 cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg
    10741 gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa
    10801 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
    10861 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
    10921 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
    10981 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
    11041 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
    11101 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
    11161 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
    11221 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
    11281 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
    11341 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
    11401 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
    11461 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    11521 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    11581 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    11641 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    11701 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    11761 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    11821 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
    11881 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
    11941 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
    12001 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
    12061 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
    12121 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
    12181 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
    12241 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
    12301 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
    12361 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
    12421 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
    12481 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
    12541 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
    12601 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
    12661 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
    12721 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
    12781 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
    12841 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
    12901 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
    12961 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
    13021 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
    13081 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
    13141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
    13201 gtgccacctg ac
//
