LOCUS       sequence-386124-       13639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tricistronic expression of Cas9-mApple-BSD in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13639)
  AUTHORS   Teh SSK, Bowland K, Halper-Stromberg E, Kotwal A, Bennett A, Skaist 
            A, Tang J, Cai F, Macoretta A, Liang H, Kamiyama H, Wheelan S, Lin 
            MT, Hruban RH, Hung CF, Goldstein M, Scharpf RB, Roberts NJ, 
            Eshleman JR
  TITLE     CRISPR-Cas9 for selective targeting of somatic mutations in 
            pancreatic cancers.
  JOURNAL   NAR Cancer. 2024 Jun 19;6(2):zcae028. doi: 10.1093/narcan/zcae028. 
            eCollection 2024 Jun.
  PUBMED    38915758
REFERENCE   2  (bases 1 to 13639)
  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: "NAR 
            Cancer."; date: "2024-06-19"; volume: "6(2):zcae028. doi"; pages: " 
            10.1093/narcan/zcae028. eCollection 2024 Jun"
COMMENT     Sequence Label: pLentiCas9-T2A-mApple
FEATURES             Location/Qualifiers
     source          1..13639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..126
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    259..483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     472..489
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             515..695
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     1200..1216
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1201..1217
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1220..1808
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1326..1337
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1735..1755
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1833..2231)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(2232..2288)
                     /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             complement(2298..2315)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2316..3023)
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2591..2610)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      3020..3029
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3036..3089)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(3099..3122)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3123..3170)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3171..7274)
                     /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             complement(3171..7274)
                     /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"
     regulatory      7271..7280
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7299..7510)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    7603..7720
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(8018..8062)
                     /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    8247..8480
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    8973..9098
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             9145..9325
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(9343..9541)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(9368..9388)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        9543..9922
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     10094..10113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        10188..10292
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10293..10361,10362..11153)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(10511..10530)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11324..11912
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11813..11832
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12066..12083
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12200..12221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12236..12241,12242..12259,12260..12266)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12274..12290
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     12279..12301
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12298..12314
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12298..12314
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    12363..12484
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12374..12393)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     12428..12447
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(12614..12988)
                     /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"
     promoter        complement(13007..13054)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     rep_origin      13116..13250
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(13170..13189)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     13395..13415
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(join(13424..13639,1..213))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13534..13555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
       61 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
      121 aaggagcggg cgctagggcg ctgacaagtg tagcggtcac gctgcgcgta accaccacac
      181 ccgccgcgct taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg
      241 ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt
      301 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
      361 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
      421 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
      481 cagtcgaggc tgatcagcgg gtttaaacgg gccctgctag agattttcca cactgactaa
      541 aagggtctga gggatctcta gttaccagag tcacacaaca gacgggcaca cactacttga
      601 agcactcaag gcaagcttta ttgaggctta agcagtgggt tccctagtta gccagagagc
      661 tcccaggctc agatctggtc taaccagaga gacccagtac agtccggatg cagctctcgg
      721 gccatgtgat gaaatgctag gcggctgtca aacctccact ctaatacttc tctctccggg
      781 tcatccatcc catgcaggct cacagggtgt aacaagcggg tgttctctcc ttcattggct
      841 tcttctacct tctcttgctc aactggtact agcttgtagc accatccaaa ggtcagtgga
      901 tatctgatcc ctggccctgg tgtgtagttc tgccaatcag ggaagtagcc ttgtgtgtgg
      961 tagatccaca gatcaaggat atcttgtctt cgttgggagt gaattagccc ttccagtccc
     1021 cccttttctt ttaaaaagtg gctaagatct acagctgcct tgtaagtcat tggtcttaaa
     1081 ggtacctgag gtgtgactgg aaaacccacc tcctcctcct cttgtgcttc tagccaggca
     1141 caatcagcat tggtagctgc tgtattgcta cttgtgattg ctccatgttt ttctaggtct
     1201 cgaggtcgac ggtatcgatg cggggaggcg gcccaaaggg agatccgact cgtctgaggg
     1261 cgaaggcgaa gacgcggaag aggccgcaga gccggcagca ggccgcggga aggaaggtcc
     1321 gctggattga gggccgaagg gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca
     1381 acacaggcga gcagccaagg aaaggacgat gatttccccg acaacaccac ggaattgtca
     1441 gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc aggcggcgat gagttccgcc
     1501 gtggcaatag ggagggggaa agcgaaagtc ccggaaagga gctgacaggt ggtggcaatg
     1561 ccccaaccag tgggggttgc gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca
     1621 acgggccaca actcctcata aagagacagc aaccaggatt tatacaagga ggagaaaatg
     1681 aaagccatac gggaagcaat agcatgatac aaaggcatta aagcagcgta tccacatagc
     1741 gtaaaaggag caacatagtt aagaatacca gtcaatcttt cacaaatttt gtaatccaga
     1801 ggttgattac cgataagctt gatatcgaat tcttagccct cccacacata accagagggc
     1861 agcaattcac gaatcccaac tgccgtcggc tgtccatcac tgtccttcac tatggctttg
     1921 atcccaggat gcagatcgag aagcacctgt cggcaccgtc cgcaggggct caagatgccc
     1981 ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc cagctgccgc agcagcagca
     2041 gtgcccagca ccacgagttc tgcacaaggt cccccagtaa aatgatatac attgacacca
     2101 gtgaagatgc ggccgtcgct agagagagct gcgctggcga cgctgtagtc ttcagagatg
     2161 gggatgctgt tgattgtagc cgttgctctt tcaatgaggg tggattcttc ttgagacaaa
     2221 ggcttggcca tcggtccagg attctcttcg acatctccgg cttgtttcag cagagagaag
     2281 tttgttgcgc cggatccgtg gtgatggtga tgatgcttgt acagctcgtc catgccgccg
     2341 gtggagtggc ggccctcggc gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg
     2401 gacacgatgt ccaacttgat gtcgacgatg taggcgccgg gcagctgcac gggcttcttg
     2461 gccttgtagg tggtcttgac ctcggcggcg tagtggccgc cgtccttcag cttcagcctc
     2521 ttcttgatct cgctcttcag ggcgccgtcc tcggggtaca tccgctcctc ggaggcctcc
     2581 cagcccatgg tcttcttctg cattacgggg ccgtcggagg ggaagttggt gccgcgcagc
     2641 ttcaccttgt agatgaacac gccgtcctgc agggaggagt cctggttaac gtgaataatg
     2701 ccgccgtcct cgaagttcat cacgcgctcc cacctgaagc cctcggggaa ggacagcttg
     2761 aagtagtcgg ggatgtcggc tgggtgctta atgtagacct tggagccgta catgaactga
     2821 ggggacagga tgtcccaggc gaagggcagg gggccaccct tggtcacctt cagcttagcg
     2881 gtctgaaagg cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg
     2941 ttcacggagc cctccatgtg caccttgaag cgcatgaact ccttgatgat ggccatgtta
     3001 ttctcctcgc ccttgctcac catggtggcg accggtgggc caggattctc ctcgacgtca
     3061 ccgcatgtta gcagacttcc tctgccctct ccggatccct tatcgtcatc gtctttgtaa
     3121 tctttcttct tcttagcctg tccagccttc tttgtggcgg caggtcgctt gtcgcctccc
     3181 agctgagaca ggtcgatccg tgtctcgtac aggccggtga tgctctggtg gatcagggtg
     3241 gcgtccagca cctctttggt gctggtgtac ctcttccggt cgatggtggt gtcaaagtac
     3301 ttgaaggcgg caggggctcc cagattggtc agggtaaaca ggtggatgat attctcggcc
     3361 tgctctctga tgggcttatc ccggtgcttg ttgtaggcgg acagcacttt gtccagatta
     3421 gcgtcggcca ggatcactct cttggagaac tcgctgatct gctcgatgat ctcgtccagg
     3481 tagtgcttgt gctgttccac aaacagctgt ttctgctcat tatcctcggg ggagcccttc
     3541 agcttctcat agtggctggc caggtacagg aagttcacat atttggaggg cagggccagt
     3601 tcgtttccct tctgcagttc gccggcagag gccagcattc tcttccggcc gttttccagc
     3661 tcgaacaggg agtacttagg cagcttgatg atcaggtcct ttttcacttc tttgtagccc
     3721 ttggcttcca gaaagtcgat gggattcttc tcgaagctgc ttctttccat gatggtgatc
     3781 cccagtagct ctttcacact cttcagtttc ttggacttgc ccttttccac tttggccacc
     3841 accagcacag aataggccac ggtggggctg tcgaagccgc cgtacttctt agggtcccag
     3901 tccttctttc tggcgatcag cttatcgctg ttcctcttgg gcaggataga ctctttgctg
     3961 aagccgcctg tctgcacctc ggtctttttc acgatattca cttggggcat gctcagcact
     4021 ttccgcacgg tggcaaaatc ccggccctta tcccacacga tctccccggt ttcgccgttt
     4081 gtctcgatca gaggccgctt ccggatctcg ccgttggcca gggtaatctc ggtcttgaaa
     4141 aagttcatga tgttgctgta gaagaagtac ttggcggtag ccttgccgat ttcctgctcg
     4201 ctcttggcga tcatcttccg cacgtcgtac accttgtagt cgccgtacac gaactcgctt
     4261 tccagcttag ggtacttttt gatcagggcg gttcccacga cggcgttcag gtaggcgtcg
     4321 tgggcgtggt ggtagttgtt gatctcgcgc actttgtaaa actggaaatc cttccggaaa
     4381 tcggacacca gcttggactt cagggtgatc actttcactt cccggatcag cttgtcattc
     4441 tcgtcgtact tagtgttcat ccgggagtcc aggatctgtg ccacgtgctt tgtgatctgc
     4501 cgggtttcca ccagctgtct cttgatgaag ccggccttat ccagttcgct caggccgcct
     4561 ctctcggcct tggtcagatt gtcgaacttt ctctgggtaa tcagcttggc gttcagcagc
     4621 tgccgccagt agttcttcat cttcttcacg acctcttcgg agggcacgtt gtcgctcttg
     4681 ccccggttct tgtcgcttct ggtcagcacc ttgttgtcga tggagtcgtc cttcagaaag
     4741 ctctgaggca cgatatggtc cacatcgtag tcggacagcc ggttgatgtc cagttcctgg
     4801 tccacgtaca tatcccgccc attctgcagg tagtacaggt acagcttctc gttctgcagc
     4861 tgggtgtttt ccacggggtg ttctttcagg atctggctgc ccagctcttt gatgccctct
     4921 tcgatccgct tcattctctc gcggctgttc ttctgtccct tctgggtggt ctggttctct
     4981 ctggccattt cgatcacgat gttctcgggc ttgtgccggc ccatcacttt cacgagctcg
     5041 tccaccacct tcactgtctg caggatgccc ttcttaatgg cggggctgcc ggccagattg
     5101 gcaatgtgct cgtgcaggct atcgccctgg ccggacacct gggctttctg gatgtcctct
     5161 ttaaaggtca ggctgtcgtc gtggatcagc tgcatgaagt ttctgttggc gaagccgtcg
     5221 gacttcagga aatccaggat tgtcttgccg gactgcttgt cccggatgcc gttgatcagc
     5281 ttccggctca gcctgcccca gccggtgtat ctccgccgct tcagctgctt catcactttg
     5341 tcgtcgaaca ggtgggcata ggttttcagc cgttcctcga tcatctctct gtcctcaaac
     5401 agtgtcaggg tcagcacgat atcttccaga atgtcctcgt tttcctcatt gtccaggaag
     5461 tccttgtcct tgataatttt cagcagatcg tggtatgtgc ccagggaggc gttgaaccga
     5521 tcttccacgc cggagatttc cacggagtcg aagcactcga ttttcttgaa gtagtcctct
     5581 ttcagctgct tcacggtcac tttccggttg gtcttgaaca gcaggtccac gatggccttt
     5641 ttctgctcgc cgctcaggaa ggcgggcttt ctcattccct cggtcacgta tttcactttg
     5701 gtcagctcgt tatacacggt gaagtactcg tacagcaggc tgtgcttggg cagcaccttc
     5761 tcgttgggca ggttcttatc gaagttggtc atccgctcga tgaagctctg ggcggaagcg
     5821 cccttgtcca ccacttcctc gaagttccag ggggtgatgg tttcctcgct ctttctggtc
     5881 atccaggcga atctgctgtt tcccctggcc agagggccca cgtagtaggg gatgcggaag
     5941 gtcaggatct tctcgatctt ttcccggttg tccttcagga atgggtaaaa atcttcctgc
     6001 cgccgcagaa tggcgtgcag ctctcccagg tggatctggt gggggatgct gccgttgtcg
     6061 aaggtccgct gcttccgcag caggtcctct ctgttcagct tcacgagcag ttcctcggtg
     6121 ccgtccatct tttccaggat gggcttgatg aacttgtaga actcttcctg gctggctccg
     6181 ccgtcaatgt agccggcgta gccgttcttg ctctggtcga agaaaatctc tttgtacttc
     6241 tcaggcagct gctgccgcac gagagctttc agcagggtca ggtcctggtg gtgctcgtcg
     6301 tatctcttga tcatagaggc gctcaggggg gccttggtga tctcggtgtt cactctcagg
     6361 atgtcgctca gcaggatggc gtcggacagg ttcttggcgg ccagaaacag gtcggcgtac
     6421 tggtcgccga tctgggccag caggttgtcc aggtcgtcgt cgtaggtgtc cttgctcagc
     6481 tgcagtttgg catcctcggc caggtcgaag ttgctcttga agttgggggt caggcccagg
     6541 ctcagggcaa tcaggtttcc gaacaggcca ttcttcttct cgccgggcag ctgggcgatc
     6601 agattttcca gccgtctgct cttgctcagt ctggcagaca ggatggcctt ggcgtccacg
     6661 ccgctggcgt tgatggggtt ttcctcgaac agctggttgt aggtctgcac cagctggatg
     6721 aacagcttgt ccacgtcgct gttgtcgggg ttcaggtcgc cctcgatcag gaagtggccc
     6781 cggaacttga tcatgtgggc cagggccaga tagatcagcc gcaggtcggc cttgtcggtg
     6841 ctgtccacca gtttctttct caggtggtag atggtggggt acttctcgtg gtaggccacc
     6901 tcgtccacga tgttgccgaa gatggggtgc cgctcgtgct tcttatcctc ttccaccagg
     6961 aaggactctt ccagtctgtg gaagaagctg tcgtccacct tggccatctc gttgctgaag
     7021 atctcttgca gatagcagat ccggttcttc cgtctggtgt atcttcttct ggcggttctc
     7081 ttcagccggg tggcctcggc tgtttcgccg ctgtcgaaca gcagggctcc gatcaggttc
     7141 ttcttgatgc tgtgccggtc ggtgttgccc agcaccttga atttcttgct gggcaccttg
     7201 tactcgtcgg tgatcacggc ccagcccaca gagttggtgc cgatgtccag gccgatgctg
     7261 tacttcttgt ccatggtggc agcgctctag aaccggtcct gtgttctggc ggcaaacccg
     7321 ttgcgaaaaa gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg
     7381 ggaaaaaggc ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc
     7441 taggcaccgg atcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg
     7501 cgctctgccc actgacgggc accggagcca attcccactc ctttcaagac ctagctagct
     7561 aattaaccaa actggatctc tgctgtccct gtaataaacc cgaaaatttt gaatttttgt
     7621 aatttgtttt tgtaattctt tagtttgtat gtctgttgct attatgtcta ctattctttc
     7681 ccctgcactg taccccccaa tccccccttt tcttttaaaa ttgtggatga atactgccat
     7741 ttgtctgcag aattggcgca cgcagtgccg atccgttcac taatcgaatg gatctgtctc
     7801 tgtctctctc tccaccttct tcttctattc cttcgggcct gtcgggtccc ctcggggttg
     7861 ggaggtgggt ctgaaacgat aatggtgaat atccctgcct aactctattc actatagaaa
     7921 gtacagcaaa aactattctt aaacctacca agcctcctac tatcattatg aataatttta
     7981 tataccacag ccaatttgtt atgttaaacc aattccacaa acttgcccat ttatctaatt
     8041 ccaataattc ttgttcattc ttttcttgct ggttttgcga ttcttcaatt aaggagtgta
     8101 ttaagcttgt gtaattgtta atttctctgt cccactccat ccaggtcgtg tgattccaaa
     8161 tctgttccag agatttatta ctccaactag cattccaagg cacagcagtg gtgcaaatga
     8221 gttttccaga gcaaccccaa atccccagga gctgttgatc ctttaggtat ctttccacag
     8281 ccaggattct tgcctggagc tgcttgatgc cccagactgt gagttgcaac agatgctgtt
     8341 gcgcctcaat agccctcagc aaattgttct gctgctgcac tataccagac aataattgtc
     8401 tggcctgtac cgtcagcgtc attgacgctg cgcccatagt gcttcctgct gctcccaaga
     8461 acccaaggaa caaagctcct attcccactg ctcttttttc tctctgcacc actcttctct
     8521 ttgccttggt gggtgctact cctaatggtt caatttttac tactttatat ttatataatt
     8581 cacttctcca attgtccctc atatctcctc ctccaggtct gaagatcagc ggccgcttgc
     8641 tgtgcggtgg tcttactttt gttttgctct tcctctatct tgtctaaagc ttccttggtg
     8701 tcttttatct ctatcctttg atgcacacaa tagagggttg ctactgtatt atataatgat
     8761 ctaagttctt ctgatcctgt ctgaagggat ggttgtagct gtcccagtat ttgtctacag
     8821 ccttctgatg tttctaacag gccaggatta actgcgaatc gttctagctc cctgcttgcc
     8881 catactatat gttttaattt atattttttc tttccccctg gccttaaccg aattttttcc
     8941 catcgcgatc taattctccc ccgcttaata ctgacgctct cgcacccatc tctctccttc
     9001 tagcctccgc tagtcaaaat ttttggcgta ctcaccagtc gccgcccctc gcctcttgcc
     9061 gtgcgcgctt cagcaagccg agtcctgcgt cgagagagct cctctggttt ccctttcgct
     9121 ttcaagtccc tgttcgggcg ccactgctag agattttcca cactgactaa aagggtctga
     9181 gggatctcta gttaccagag tcacacaaca gacgggcaca cactacttga agcactcaag
     9241 gcaagcttta ttgaggctta agcagtgggt tccctagtta gccagagagc tcccaggctc
     9301 agatctggtc taaccagaga gacccagtac aggcaaaacg cgctgcttat atagacctcc
     9361 caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga
     9421 aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt
     9481 ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc
     9541 accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag
     9601 gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc
     9661 gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc
     9721 acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt
     9781 gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat
     9841 gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga ttactattaa
     9901 taactagtca ataatcaatg tcaacgcgta tatctggccc gtacatcgcg aagcagcgca
     9961 aaacgcctaa ccctaagcag attcttcatg caattgtcgg tcaagccttg ccttgttgta
    10021 gcttaaattt tgctcgcgca ctactcagcg acctccaaca cacaagcagg gagcagatac
    10081 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag gggatcggga
    10141 gatctcccga tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
    10201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
    10261 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
    10321 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
    10381 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
    10441 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
    10501 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
    10561 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
    10621 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
    10681 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
    10741 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
    10801 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    10861 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    10921 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    10981 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    11041 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    11101 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    11161 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    11221 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    11281 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    11341 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    11401 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    11461 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    11521 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    11581 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    11641 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    11701 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    11761 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    11821 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    11881 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    11941 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    12001 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    12061 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
    12121 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
    12181 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
    12241 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
    12301 gaaacagcta tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat
    12361 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
    12421 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
    12481 agttggggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg
    12541 cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat
    12601 ctcgtagcac gtgtcagtcc tgctcctcgg ccacgaagtg cacgcagttg ccggccgggt
    12661 cgcgcagggc gaactcccgc ccccacggct gctcgccgat ctcggtcatg gccggcccgg
    12721 aggcgtcccg gaagttcgtg gacacgacct ccgaccactc ggcgtacagc tcgtccaggc
    12781 cgcgcaccca cacccaggcc agggtgttgt ccggcaccac ctggtcctgg accgcgctga
    12841 tgaacagggt cacgtcgtcc cggaccacac cggcgaagtc gtcctccacg aagtcccggg
    12901 agaacccgag ccggtcggtc cagaactcga ccgctccggc gacgtcgcgc gcggtgagca
    12961 ccggaacggc actggtcaac ttggccatgg tttagttcct caccttgtcg tattatacta
    13021 tgccgatata ctatgccgat gattaattgt caacacgtgc tgatcagatc cgaaaatgga
    13081 tatacaagct cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
    13141 cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag
    13201 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga ctatggttgc
    13261 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
    13321 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
    13381 tggggacttt ccacacccta actgacacac attccacaga attaattcgc gttaaatttt
    13441 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
    13501 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
    13561 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
    13621 cgtgaaccat caccctaat
//
