LOCUS       sequence-350708-       14744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS414 backbone containing LINEAR fragment targeting BAT1 in Y. 
            lipolytica.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14744)
  AUTHORS   Ploessl D, Zhao Y, Cao M, Ghosh S, Lopez C, Sayadi M, Chudalayandi 
            S, Severin A, Huang L, Gustafson M, Shao Z
  TITLE     A repackaged CRISPR platform increases homology-directed repair for 
            yeast engineering.
  JOURNAL   Nat Chem Biol. 2021 Oct 28. pii: 10.1038/s41589-021-00893-5. doi: 
            10.1038/s41589-021-00893-5.
  PUBMED    34711982
REFERENCE   2  (bases 1 to 14744)
  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: "Nat Chem 
            Biol. 2021 Oct 28. pii: 10.1038/s41589-021-00893-5. doi: 
            10.1038/s41589-021-00893-5."
COMMENT     Sequence Label: YL-CashGem-BAT1-URA3-LINEAR
FEATURES             Location/Qualifiers
     source          1..14744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..33)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(15..33)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(43..60)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(43..59)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(52..74)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      200..655
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(287..306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     497..518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(758..1432)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(1433..1713)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(1734..1753)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1853..1875
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1913..1931)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1972..2475
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        2512..2616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2617..2685,2686..3477)
                     /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(2835..2854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3648..4236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4137..4156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4390..4407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4524..4545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4560..4565,4566..4583,4584..4590)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4598..4614
                     /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     4603..4625
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4622..4638
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4622..4638
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4657..4677
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4659..4677
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4851..4869
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(4918..4938)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4951..5280)
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     CDS             complement(5326..9429)
                     /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(5326..9429)
                     /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(5326..9429)
                     /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"
     ncRNA           10934..10970
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        10991..11066
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           11071..11138
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(12139..12172)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(14211..14243)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
       61 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
      121 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
      181 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
      241 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
      301 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
      361 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
      421 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
      481 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
      541 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
      601 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
      661 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagat cggcaagtgc
      721 acaaacaata cttaaataaa tactactcag taataaccta tttcttagca tttttgacga
      781 aatttgctat tttgttagag tcttttacac catttgtctc cacacctccg cttacatcaa
      841 caccaataac gccatttaat ctaagcgcat caccaacatt ttctggcgtc agtccaccag
      901 ctaacataaa atgtaagctt tcggggctct cttgccttcc aacccagtca gaaatcgagt
      961 tccaatccaa aagttcacct gtcccacctg cttctgaatc aaacaaggga ataaacgaat
     1021 gaggtttctg tgaagctgca ctgagtagta tgttgcagtc ttttggaaat acgagtcttt
     1081 taataactgg caaaccgagg aactcttggt attcttgcca cgactcatct ccatgcagtt
     1141 ggacgatatc aatgccgtaa tcattgacca gagccaaaac atcctcctta ggttgattac
     1201 gaaacacgcc aaccaagtat ttcggagtgc ctgaactatt tttatatgct tttacaagac
     1261 ttgaaatttt ccttgcaata accgggtcaa ttgttctctt tctattgggc acacatataa
     1321 tacccagcaa gtcagcatcg gaatctagag cacattctgc ggcctctgtg ctctgcaagc
     1381 cgcaaacttt caccaatgga ccagaactac ctgtgaaatt aataacagac atactccaag
     1441 ctgcctttgt gtgcttaatc acgtatactc acgtgctcaa tagtcaccaa tgccctccct
     1501 cttggccctc tccttttctt ttttcgaccg aattaattct taatcggcaa aaaaagaaaa
     1561 gctccggatc aagattgtac gtaaggtgac aagctatttt tcaataaaga atatcttcca
     1621 ctactgccat ctggcgtcat aactgcaaag tacacatata ttacgatgct gttctattaa
     1681 atgcttccta tattatatat atagtaatgt cgtgatctat ggtgcactct cagtacaatc
     1741 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     1801 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     1861 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     1921 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac ggatcgcttg
     1981 cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg
     2041 tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag
     2101 agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta
     2161 ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac
     2221 gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga
     2281 aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg
     2341 cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct
     2401 ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat tataattatt
     2461 tttatagcac gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc
     2521 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2581 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2641 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2701 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     2761 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2821 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     2881 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     2941 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3001 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3061 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3121 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3181 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3241 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3301 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3361 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3421 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3481 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3541 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3601 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3661 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3721 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3781 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3841 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3901 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3961 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4021 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4081 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4141 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4201 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4261 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4321 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4381 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     4441 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4501 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     4561 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     4621 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
     4681 caaaagctgg agctccaccg cggtggcggc cgcctgtaca gaaaaaaaag aaaaatttga
     4741 aatataaata acgttcttaa tactaacata actataaaaa aataaatagg gacctagact
     4801 tcaggttgtc taactccttc cttttcggtt agagcggatg tggggggagg gcgtgaatgt
     4861 aagcgtgaca taactaatta catgactcga ggtccaatga ctcgaggtcc aaaactacac
     4921 cttcctcttc ttcttggggt cagccctgct caaagccttt cttctctttt cagcaacctc
     4981 tttccagtac tgagaagaag ggttctcttt gatcatcaag tcgaaagatt cttgagtgac
     5041 accaccaagg ttcttgtttt cagaagattc tggaacgata acacctggag aagaggtagt
     5101 agaagtcaag tggtcgtttc tatgctttct cttagacaaa ccagcagaca attcgttttc
     5161 tctaccaacc aaagaaccag aggcagatgg ttggatcatc ttcaaagttc ttctagggac
     5221 agacgagttc ttgatgtttt ctttgatctc ttcttgcttc tgcttcatag atgggttcat
     5281 cgaccctccg cctccgctac cgcctccacc agagcctcct ccaccgtctc caccgagctg
     5341 agagaggtcg attcttgttt catagagccc cgtaattgac tgatgaatca gtgtggcgtc
     5401 caggacctcc tttgtagagg tgtaccgctt tctgtctatg gtggtgtcga agtacttgaa
     5461 ggctgcaggc gcgcccaagt tggtcagagt aaacaagtgg ataatgtttt ctgcctgctc
     5521 cctgatgggc ttatccctgt gcttattgta agcagaaagc accttatcga ggttagcgtc
     5581 ggcgaggatc actcttttgg agaattcgct tatttgctcg atgatctcat caaggtagtg
     5641 tttgtgttgt tccacgaaca gctgcttctg ctcattatct tcgggagacc ctttgagctt
     5701 ttcatagtgg ctggccagat acaagaaatt aacgtattta gagggcagtg ccagctcgtt
     5761 acctttctgc agctcgcccg cactagcgag cattcgtttc cggccgtttt caagctcaaa
     5821 gagagagtac ttgggaagct taatgatgag gtcttttttg acctctttat atcctttcgc
     5881 ctcgagaaag tcgatggggt ttttttcgaa gcttgatcgc tccatgattg tgatgcccag
     5941 cagttccttg acgcttttga gttttttaga cttccctttc tccactttgg ccacaaccag
     6001 tacactgtaa gcgactgtag gagaatcgaa tccgccgtat ttcttggggt cccaatcttt
     6061 tttgcgtgcg atcagcttgt cgctgttcct tttcgggagg atactttcct tggagaagcc
     6121 tccggtctgt acttcggtct ttttaacgat gttcacctgc ggcatggaca ggaccttccg
     6181 gactgtcgcg aaatccctac ccttgtccca cacgatttct cctgtttctc cgtttgtttc
     6241 gataagtggt cgcttccgaa tctctccatt ggccagtgta atctcggtct tgaaaaaatt
     6301 cataatattg ctgtaaaaga agtacttagc ggtggccttg cctatttcct gctcagactt
     6361 tgcgatcatt ttcctaacat cgtacacttt atagtctccg taaacaaatt cagattcaag
     6421 cttgggatat tttttgataa gtgcagtgcc taccactgca ttcaggtagg catcatgcgc
     6481 atggtggtaa ttgttgatct ctctcacctt ataaaactga aagtcctttc tgaaatctga
     6541 gaccagctta gacttcagag taataacttt cacctctcga atcagtttgt cattttcatc
     6601 gtacttggtg ttcatgcgtg aatcgagaat ttgggccacg tgcttggtga tctggcgtgt
     6661 ctcaacaagc tgccttttga tgaagccggc tttatccaac tcagacaggc cacctcgttc
     6721 agccttagtc agattatcga acttccgttg tgtgatcagt ttggcgttca gcagctgccg
     6781 ccaataattt ttcattttct tgacaacttc ttctgagggg acgttatcac tcttccctct
     6841 atttttatcg gatcttgtca acactttatt atcaatagaa tcatctttga gaaaagactg
     6901 gggcacgata tgatccacgt cgtagtcgga gagccgattg atgtccagtt cctgatccac
     6961 gtacatgtcc ctgccgttct gcaggtagta caggtagagc ttctcattct gaagctgggt
     7021 gttttcaact gggtgttcct taaggatttg ggaccccagt tcttttatac cctcttcaat
     7081 cctcttcatc ctttccctac tgttcttctg tcccttctgg gtagtttggt tctctcgggc
     7141 catctcgata acgatattct cgggcttatg ccttcccatt actttgacga gttcatccac
     7201 gaccttaacg gtctgcagta ttcccttttt gatagctggg ctacctgcaa gattagcgat
     7261 gtgctcgtga agactgtccc cctggccaga aacttgtgct ttctggatgt cctccttaaa
     7321 ggtgagagag tcatcatgga tcaactgcat gaagttccgg ttggcaaatc catcggactt
     7381 aagaaaatcc aggattgtct ttccactctg cttgtctcgg atcccattga tcagttttct
     7441 tgacagccgc ccccatcctg tatatcggcg cctcttgagc tgtttcatga ctttgtcgtc
     7501 gaagagatga gcgtaagttt tcaagcgttc ttcaatcatc tccctatctt caaacaacgt
     7561 aagggtgagg acaatgtcct caagaatgtc ctcgttctcc tcattgtcca ggaagtcctt
     7621 gtctttaatg attttcagga gatcgtgata cgttcccagg gatgcgttga agcgatcctc
     7681 cactccgctg atttcaacag agtcgaaaca ttcaatcttt ttgaaatagt cttctttgag
     7741 ctgtttcacg gtaactttcc ggttcgtctt gaagaggagg tccacgatag ctttcttctg
     7801 ctctccagac aggaatgctg gctttctcat cccttctgtg acgtatttga ccttggtgag
     7861 ctcgttataa actgtgaagt actcgtacag cagagagtgt ttaggaagca ccttttcgtt
     7921 aggcagattt ttatcaaagt tagtcatcct ttcgatgaag gactgggcag aggccccctt
     7981 atccacgact tcctcgaagt tccagggagt gatggtctct tctgatttgc gagtcatcca
     8041 cgcgaatctg gaatttcccc gggcgagggg gcctacatag tagggtatcc gaaatgtgag
     8101 gattttctca atcttttccc tgttatcttt caaaaagggg tagaaatcct cttgccgcct
     8161 gaggatagcg tgcagttcgc ccaggtgaat ctggtggggg atgcttccat tgtcgaaagt
     8221 gcgctgtttg cgcaacagat cttctctgtt aagctttacc agcagctcct cggtgccgtc
     8281 cattttttcc aagatgggct taataaattt gtaaaattcc tcctggcttg ctccgccgtc
     8341 aatgtatccg gcgtagccat ttttagactg atcgaagaaa atttccttgt acttctcagg
     8401 cagttgctgt ctgacaaggg ccttcagcaa agtcaagtct tggtggtgct catcatagcg
     8461 cttgatcata ctagcgctca gcggagcttt ggtgatctcc gtgttcactc gcagaatatc
     8521 actcagcaga atggcgtctg acaggttctt tgccgccaaa aaaaggtctg cgtactggtc
     8581 gccgatctgg gccagcagat tgtcgagatc atcatcgtag gtgtctttgc tcagttgaag
     8641 cttggcatct tcggccaggt cgaagttaga tttaaagttg ggggtcagcc cgagtgacag
     8701 ggcgataaga ttaccaaaca ggccgttctt cttctcccca gggagctgtg cgatgaggtt
     8761 ttcgagccgc cgggatttgg acagcctagc gctcaggatt gctttggcgt caactccgga
     8821 tgcgttgatc gggttctctt cgaaaagctg attgtaagtc tgaaccagtt ggataaagag
     8881 tttgtcaaca tcgctgttgt ctgggttcag gtccccctcg atgaggaagt gtccccgaaa
     8941 tttgatcata tgcgccagcg cgagatagat caaccgcaag tcagccttat cagtactgtc
     9001 tacaagcttc ttcctcagat gatatatggt tgggtacttt tcatggtacg ccacctcgtc
     9061 cacgatattg ccaaagattg ggtggcgctc gtgcttttta tcctcctcca ccaaaaagga
     9121 ctcctccagc ctatggaaga aagagtcatc caccttagcc atctcattac taaagatctc
     9181 ctgcaggtag cagatccgat tctttctgcg ggtatatctg cgccgtgctg ttcttttgag
     9241 ccgcgtggct tcggccgtct ccccggagtc gaacaggagg gcgccaatga ggttcttctt
     9301 tatgctgtgg cgatcggtat tgcccagaac tttgaatttt ttgctcggca ccttgtactc
     9361 gtccgtaatg acggcccaac cgacgctgtt tgtgccgata tcgagcccaa tggagtactt
     9421 cttgtccatt gttgatgtgt gtttaattca agaatgaata tagagtgttg atgtgtgttt
     9481 aattcaagaa tgaatataga gaagagaaga agaaaaaaga ttcaattgag ccggcgatgc
     9541 agacccttat ataaatgttg ccttggacag acggagcaag cccgcccaaa cctacgttcg
     9601 gtataatatg ttaagctttt taacacaaag gtttggcttg gggtaacctg atgtggtgca
     9661 aaagaccggg cgttggcgag ccattgcgcg ggcgaatggg gccgtgactc gtctcaaatt
     9721 cgagggcgtg cctcaattcg tgcccccgtg gctttttccc gccgtttccg ccccgtttgc
     9781 accactgcag ccgcttcttt ggttcggaca ccttgctgcg agctaggtgc cttgtgctac
     9841 ttaaaaagtg gcctcccaac accaacatga catgagtgcg tgggccaaga cacgttggcg
     9901 gggtcgcagt cggctcaatg gcccggaaaa aacgctgctg gagctggttc ggacgcagtc
     9961 cgccgcggcg tatcgatatc cgcaaggttc catggcgcca ttgccctccg tcggcgtcta
    10021 tcccgcaacc tctaaataga gcgggaatat aacccaagct tctttttttt tcctttaaca
    10081 cgcacacccc caactatcat gttgctgctg ctgtttgact ctactctgtg gaggggtgct
    10141 cccacccaac ccaacctaca ggtggatccg gcgctgtgat tggctgataa gtctcctatc
    10201 cggactaatt ctgaccaatg ggacatgcgc gcaggaccca aatgccgcaa ttacgtaacc
    10261 ccaacgaaat gcctacccct ctttggagcc cagcggcccc aaatcccccc aagcagcccg
    10321 gttctaccgg cttccatctc caagcacccc tttctccaca ccccacaaaa agacccgtgc
    10381 aggacatcct actgcgagag accgggttgg cggcgtattt gtgtcccaaa aaacagcccc
    10441 aattgcccca attgacccca aattgaccca gtagcgggcc caaccccggc gagagccccc
    10501 ttcaccccac atatcaaacc tcccccggtt cccacacttg ccgttaaggg cgtagggtac
    10561 tgcagtctgg aatctacgct tgttcagact ttgtactagt ttctttgtct ggccatccgg
    10621 gtaacccatg ccggacgcaa aatagactac tgaaaatttt tttgctttgt ggttgggact
    10681 ttagccaagg gtataaaaga ccaccgtccc cgaattacct ttcctcttct tttctctctc
    10741 tccttgtcaa ctcacacccg aaatcgttaa gcatttcctt ctgagtataa gaatcattca
    10801 aaatggtgag tttcagaggc agcagcaatt gccacgggct ttgagcacac ggccgggtgt
    10861 ggtcccattc ccatcgacac aagacgccac gtcatccgac cagcactttt tgcagtacta
    10921 accgcagtgc atgctgatga gtccgtgagg acgaaacgag taagctcgtc catgcaccga
    10981 atgaacacat gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
    11041 ttgaaaaagt ggcaccgagt cggtgctttt ggccggcatg gtcccagcct cctcgctggc
    11101 gccggctggg caacatgctt cggcatggcg aatgggacca ctggccggtc gataatttaa
    11161 cgtgctgagc tcagcacacg cattgcccat tggctgtata tagatgaatg taatgatacc
    11221 gtaagagaat gagagcacgg tattgtatta caggggatta agtacacatt acttggagtt
    11281 ctgtaccaga agacactact atacatggta tcacttacat tagagtcggt gaccgtattc
    11341 gtctcgtata gacataatat tttcctaccc cacattgttc ctgggccttc ggagcacatc
    11401 tacagtgagt gactgtttca gttgagcttg aggggttaag taagtggggg aagggtttgc
    11461 gattctgaaa aagagcatga ctaatctctc tgtggaggag caatgaagtc acgtgatgca
    11521 atcataccgg tgtatcggat ctgcctgggt gtctgattac taatcattta ctcacatgtt
    11581 ttccccagct atctcatcca tctcagagcc tcggaccagc cttcggccct tttgggtttg
    11641 atctggtgaa gaggttggaa gaggataagg cgctgactca atatgacaga gctaccttct
    11701 atcttcctgg agctcaagga tatacggact attcgttcta tgacgagaca gagtgaaaag
    11761 aattgaccaa tgggctgatt gataccaagg gcagtactag atagataata gaaaatatgt
    11821 aagcaatgtc agtaaccaag agtgagtatg ggtaactatc ataattggag acaccatcgg
    11881 cgccttatgt atcaattcat ccatttttac ttgccgctgt gctcacagcg aagagctcat
    11941 agaacattcc aacactctag taatgcacat ccaccatcca caatttgcta acaaacatca
    12001 aacattttat acataattct actggaggcg acaggacgcg acgcggagcg taaaacgccc
    12061 cacccggggt ctaacacgat gcagtagctc caaagcgaag tacaagtaat ggagttgcgt
    12121 ctaatctcgc ttctctcgat aacttcgtat agcatacatt atacgaagtt ataagaatgt
    12181 cttcagtgca aatttgaagt cctgtacttg ttggagatgg tgtctgtttt ctacgtgtag
    12241 tttccagatt gacttctcca acttttcgat gtcccgtttc agctttagag cttcgtcgac
    12301 aaaggcctgt ttctcggtgt acagagcttg gtcctccttg aagttgcgac acatgtcttg
    12361 atagtatctt ggcttctctc tcttgagctt ttccataaca agttcttctg cctccaggaa
    12421 gtccatgggt ggtttgatca tggttttggt gtagtggtag tgcagtggtg gtattgtgac
    12481 tggggatgta gttgagaata agtcatacac aagtcagctt tcttcaagcc tcatataagt
    12541 ataagtagtt caatcgtatt agctacttgt acccagcatc tccgtatcga gaaacacaac
    12601 aacatgcccc attggacaga tcatgcggat acacaggttg tgcagtatca tacatactcg
    12661 atcagacagg tcgtctgacc atcatacaag ctgaacaagc gctccatact tgcacgctct
    12721 ctatatacac agttaaatta catatccata gtctaacctc taacagttaa tcttctggta
    12781 agcctcccag ccagccttct ggtatcgctt ggcctcctca ataggatctc ggttctggcc
    12841 gtacagacct cggccgacaa ttatgatatc cgttccggta gacatgacat cctcaacagt
    12901 tcggtactgc tgtccgagag cgtctccctt gtcgtcaaga cccaccccgg gggtcagaat
    12961 aagccagtcc tcagagtcgc ccttaggtcg gttctgggca atgaagccaa ccacaaactc
    13021 ggggtcggat cgggcaagct caatggtctg cttggagtac tcgccagtgg ccagagagcc
    13081 cttgcaagac agctcggcca gcatgagcag acctctggcc agcttctcgt tgggagaggg
    13141 gaccaggaac tccttgtact gggagttctc gtagtcagag acgtcctcct tcttctgttc
    13201 agagacagtt tcctcggcac cagctcgcag gccagcaatg attccggttc cgggtacacc
    13261 gtgggcgttg gtgatatcgg accactcggc gattcggtag acaccgttct tgtactggtg
    13321 cttgacagtg ttgccaatat ctgcgaactt tctgtcctcg aacaggaaga aaccgtgctt
    13381 aagagcaagt tccttgaggg ggagcacagt gccggcgtag gtgaagtcgt caatgatgtc
    13441 gatatgggtc ttgatcatgc acacataagg tccgacctta tcggcaagct caatgagctc
    13501 cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg ccacgagctt
    13561 gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt aggagggcat
    13621 tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta tcggaacctt
    13681 atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa cttatagata
    13741 gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc tctgggcgtc
    13801 gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc agctgatatt
    13861 gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca acgaagaatg
    13921 tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag gcggcaatga
    13981 cgagtcagac agatactcgt cgaccttttc cttgggaacc accaccgtca gcccttctga
    14041 ctcacgtatt gtagccaccg acacaggcaa cagtccgtgg atagcagaat atgtcttgtc
    14101 ggtccatttc tcaccaactt taggcgtcaa gtgaatgttg cagaagaagt atgtgccttc
    14161 attgagaatc ggtgttgctg atttcaataa agtcttgaga tcagtttggc taacttcgta
    14221 tagcatacat tatacgaagt tataggtaac ggcttgggtg gtgttgtggg aagagagatc
    14281 aggtgtgagt aatttagacg ctggaggttt acacgagggg gtcgatagcc ggattttagg
    14341 gttggggtag caccagtaaa ctgatcggtg cggtatcggc gtggcccttt ttagaaccgt
    14401 ttcagacccc caagagaaaa catgaggtta tagaagtaat tagaaacgtg tattacatgg
    14461 aattccatct tcatacggcc taaatcacgt atctatgtca ttctatctga cgctctgttc
    14521 ctaacgtgtt gcaacttcaa gtctgagttt atgcaacccc ctcaacatcc agactggggt
    14581 aatagcagca gaaaaaatat tggccgatcc gacaaaaaac agagttattg agtgacaaaa
    14641 aaagctactt gaacaagtat tgtacatgta gaaaacttgt ggaaaaacga agttgagaaa
    14701 aatcatctaa ggacgtatga ataataacta actcgttgac atga
//
