LOCUS       sequence-345956-       13647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS414 backbone containing LINEAR fragment targeting ADE2 in H. 
            polymorpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13647)
  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 13647)
  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: HP-Cas9hGem-Ade2-LEU2-LINEAR
FEATURES             Location/Qualifiers
     source          1..13647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        191..471
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             472..1146
                     /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"
     rep_origin      complement(1249..1704)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1386..1407)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1598..1617
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1830..1852
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1844..1861
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1845..1861
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1871..1890
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1871..1889
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2294..2317
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2321..2341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2342..6442
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTYKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSITHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6446..6466
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6500..6829
                     /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"
     promoter        7748..8414
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     8284..8307
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     ncRNA           8421..8457
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        8478..8553
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           8558..8625
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(8667..8685)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(10874..10894)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10874..10892)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10913..10929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10913..10929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10926..10948)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10937..10953
                     /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(10961..10967,10968..10985,10986..10991))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11006..11027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11144..11161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11315..11903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11395..11414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12074..12865,12866..12934))
                     /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     12697..12716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12935..13039)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    13076..13579
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     13620..13638
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatagatc acgacattac tatatatata atataggaag catttaatag aacagcatcg
      241 taatatatgt gtactttgca gttatgacgc cagatggcag tagtggaaga tattctttat
      301 tgaaaaatag cttgtcacct tacgtacaat cttgatccgg agcttttctt tttttgccga
      361 ttaagaatta attcggtcga aaaaagaaaa ggagagggcc aagagggagg gcattggtga
      421 ctattgagca cgtgagtata cgtgattaag cacacaaagg cagcttggag tatgtctgtt
      481 attaatttca caggtagttc tggtccattg gtgaaagttt gcggcttgca gagcacagag
      541 gccgcagaat gtgctctaga ttccgatgct gacttgctgg gtattatatg tgtgcccaat
      601 agaaagagaa caattgaccc ggttattgca aggaaaattt caagtcttgt aaaagcatat
      661 aaaaatagtt caggcactcc gaaatacttg gttggcgtgt ttcgtaatca acctaaggag
      721 gatgttttgg ctctggtcaa tgattacggc attgatatcg tccaactgca tggagatgag
      781 tcgtggcaag aataccaaga gttcctcggt ttgccagtta ttaaaagact cgtatttcca
      841 aaagactgca acatactact cagtgcagct tcacagaaac ctcattcgtt tattcccttg
      901 tttgattcag aagcaggtgg gacaggtgaa cttttggatt ggaactcgat ttctgactgg
      961 gttggaaggc aagagagccc cgaaagctta cattttatgt tagctggtgg actgacgcca
     1021 gaaaatgttg gtgatgcgct tagattaaat ggcgttattg gtgttgatgt aagcggaggt
     1081 gtggagacaa atggtgtaaa agactctaac aaaatagcaa atttcgtcaa aaatgctaag
     1141 aaataggtta ttactgagta gtatttattt aagtattgtt tgtgcacttg ccgatctatg
     1201 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg
     1261 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     1321 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     1381 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     1441 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     1501 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     1561 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     1621 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     1681 ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg
     1741 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     1801 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     1861 tgagcgcgcg taatacgact cactataggg cgaattgggt accctgcagc ggccgcgagc
     1921 tctcgagaac cctcaccttt cgagaggacg atgcccgtgt ctaaatgatt cgaccagcct
     1981 aagaatgttc aacaatctat aatcagtcca tagtcaacaa gagccctcaa agttgatatc
     2041 gtttagtgat ctccctcgag atgagcggag gatggttgct ttttggggag ccctaagtgg
     2101 tacgtgcggc gagcgggatg tttccccatt aggcaaagga cgaccgggta acccctcgag
     2161 aaaaaaaatt tttcgccgct aatctggtgt tatataaagc tccccctgct cctggaattt
     2221 ttttccttgt caactcacac cggaaatcaa ggcatttcat tctgagtagt tcttaaaaac
     2281 ataatcaaca atggattata aagatgacga tgacaaacct ccaaaaaaga agagaaaggt
     2341 cgataagaaa tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat
     2401 cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca
     2461 cagtatcaaa aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc
     2521 gactcgtctc aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta
     2581 tctacaggag attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact
     2641 tgaagagtct tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa
     2701 tatagtagat gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa
     2761 attggtagat tctacttata aagcggattt gcgcttaatc tatttggcct tagcgcatat
     2821 gattaagttt cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt
     2881 ggacaaacta tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat
     2941 taacgcaagt ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg
     3001 attagaaaat ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct
     3061 cattgctttg tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga
     3121 tgctaaatta cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca
     3181 aattggagat caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt
     3241 actttcagat atcctaagag taaatactga aataactaag gctcccctat cagcttcaat
     3301 gattaaacgc tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca
     3361 acaacttcca gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg
     3421 ttatattgat gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga
     3481 aaaaatggat ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa
     3541 gcaacggacc tttgacaacg gctctattac ccatcaaatt cacttgggtg agctgcatgc
     3601 tattttgaga agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga
     3661 aaaaatcttg acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg
     3721 ttttgcatgg atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt
     3781 tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa
     3841 tcttccaaat gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta
     3901 taacgaattg acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc
     3961 aggtgaacag aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt
     4021 taagcaatta aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc
     4081 aggagttgaa gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat
     4141 taaagataaa gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt
     4201 aacattgacc ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca
     4261 cctctttgat gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg
     4321 tttgtctcga aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga
     4381 ttttttgaaa tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag
     4441 tttgacattt aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca
     4501 tgaacatatt gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt
     4561 aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat
     4621 tgaaatggca cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat
     4681 gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt
     4741 tgaaaatact caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga
     4801 catgtatgtg gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat
     4861 tgttccacaa agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga
     4921 taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa
     4981 ctattggaga caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac
     5041 gaaagctgaa cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt
     5101 ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac
     5161 taaatacgat gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa
     5221 attagtttct gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta
     5281 ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata
     5341 tccaaaactt gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat
     5401 gattgctaag tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa
     5461 tatcatgaac ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc
     5521 tctaatcgaa actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc
     5581 cacagtgcgc aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca
     5641 gacaggcgga ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc
     5701 tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta
     5761 ttcagtccta gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa
     5821 agagttacta gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt
     5881 tttagaagct aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata
     5941 tagtcttttt gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca
     6001 aaaaggaaat gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca
     6061 ttatgaaaag ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca
     6121 gcataagcat tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat
     6181 tttagcagat gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc
     6241 aatacgtgaa caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc
     6301 cgctgctttt aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga
     6361 agttttagat gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga
     6421 tttgagtcag ctaggaggtg accctccaaa aaagaagaga aaggtcggta gttccaagct
     6481 tggcggcagc ggcggcagca tgaacccatc gatgaagcag aagcaggagg agatcaagga
     6541 gaacatcaag aactcgtcgg tgccaagaag aaccctgaag atgatccagc catcggcctc
     6601 gggctcgctg gtgggcagag agaacgagct gtcggccggc ctgtcgaaga gaaagcacag
     6661 aaacgaccac ctgacctcga ccacctcgtc gccaggcgtg atcgtgccag agtcgtcgga
     6721 gaacaagaac ctgggcggcg tgacccagga gtcgttcgac ctgatgatca aggagaaccc
     6781 atcgtcgcag tactggaagg aggtggccga gaagagaaga aaggccctgt gagcggtttt
     6841 gtataactaa ataatattgg aaactaaata cgaataccca aattttttat ctaaattttg
     6901 ccgaaagatt aaaatctgca gagatatccg aaacaggtaa atggatgttt caatccctgt
     6961 agtcagtcag gaacccatat tatattacag tattagtcgc cgcttaggca cgcctttaat
     7021 tagcaaaatc aaaccttaag tgcatatgcc gtataaggga aactcaaaga actggcatcg
     7081 caaaaatgaa aacatacgtt gaaactacgg caaaggattg gtcagatcgc ttcatacagg
     7141 gaaagttcgg catcaacatc tttggataat atcagaatga gaaagaacag atacgcagta
     7201 cgttttttgg tgagctcttt gcacttcttt agttctttcc atcaatatca gttttttaaa
     7261 cttttaggac taaaagtgat gtttaacttc aaaatgttta aaattttgtt cttcccgacg
     7321 ttcattaaga atactaatac actttaataa ttattttaat attttgttct aaataatgac
     7381 ttttaattaa aaaagataaa atataaaaac atcataataa ctcaccagag gttaagaaca
     7441 aaaaaacaaa ttagatatct gctaatccaa tatagttaaa tcaatctttc cttggtataa
     7501 tgggtatatt acatatattt caaggaccga cactcctacc aaatatctaa aatttaccat
     7561 attaacataa catgtatata aacgtcaaat cataatcagc actaacgtct cacggatcgt
     7621 atatgccgta gcgacaatct aagaactatg cgaggacacg ctagggccaa ggcaaaaaga
     7681 ttccttgatt acgtaaggga gttagaatca ttttgaataa aaaacacgct ttttcagttc
     7741 gagtttatca ttatcaatac tgccatttca aagaatacgt aaataattaa tagtagtgat
     7801 tttcctaact ttatttagtc aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc
     7861 ccaaaatagg gggcgggtta cacagaatat ataacatcgt aggtgtctgg gtgaacagtt
     7921 tattcctggc atccactaaa tataatggag cccgcttttt aagctggcat ccagaaaaaa
     7981 aaagaatccc agcaccaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc
     8041 tcttagcgca actacagaga acaggggcac aaacaggcaa aaaacgggca caacctcaat
     8101 ggagtgatgc aacctgcctg gagtaaatga tgacacaagg caattgaccc acgcatgtat
     8161 ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaagctga
     8221 aaaaaaaggt tgaaaccagt tccctgaaat tattccccta cttgactaat aagtatataa
     8281 agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt aaattctact
     8341 tttatagtta gtcttttttt tagttttaaa acaccaagaa cttagtttcg aataaacaca
     8401 cataaacaaa caaattccag ctgatgagtc cgtgaggacg aaacgagtaa gctcgtcctg
     8461 gaattgatct gcttggcgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     8521 tatcaacttg aaaaagtggc accgagtcgg tgcttttggc cggcatggtc ccagcctcct
     8581 cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacacagg ccccttttcc
     8641 tttgtcgata tcatgtaatt agttatgtca cgcttacatt cacgctcgac gcctcctgat
     8701 gtgcactcaa ttgcaaccct cttcaaaaga tacctgcgaa cactgaaagt gacggtgatc
     8761 ccagaagaag aggccaagga gcttatgtca ctcacactgg cggcagacca cggggcttcc
     8821 gtgccaaagg tgaaggaaac actgcaaaaa ttgccaactt tgaacttcga cgtcctatac
     8881 gtgctattca gatacttcca gcaggtgctc aaacacaaag agctcaacaa gatgagtatt
     8941 ggcgcgctga gcgtgttgat ggctcctaac ctcactcctt ttgacggcgc caaggagatt
     9001 tgctcggaac tgctaactaa ttacaagtac tacttcgagg acggcgaaat ggtggtgcgc
     9061 tagtgctccc atatctagcg ttacgtaatc ggccctattt gatagataat aatatccaag
     9121 tcagaaaaat aaaatattaa tttaatttaa tttaggagta agtggatggt ttttcaattt
     9181 ttcccggtac cggcgcaaaa tagttgcatc attttgcaat catgagtaag aacattgtgc
     9241 ttctccctgg tgatcacgtg ggccccgagg ttgttgcgga ggccgtcaag gttctcgagg
     9301 ctgtctcgtc ggcaatcggc gttaagttca acttttccaa gcacctgatc ggcggtgcct
     9361 cgatcgatgc ttatggggtg ccattgtccg acgaggccct cgaagccgcc aagaaggctg
     9421 acgccgtttt gcttggagcc gtcggaggac ctaagtgggg aaccggctcc gtgcgtcctg
     9481 agcagggtct gttgaagatc agaaaagagc tcaacttgta cgcgaacctg cgtccatgca
     9541 gttttgcttc cgacgctctt ctgaagctat ctccactgaa atcagaaatc gtcaagggca
     9601 ctgactttgt tgttgtgcgt gagttggttg gtggaatcta ctttggtgac cgcaaggagg
     9661 atgccggcga cggagttgcc agcgacactg agagctattc tgttccagag gtgcagagaa
     9721 tcacgagaat ggcggccttt ttggcgctgc agagcgaccc accgctccca ctgtggtcgc
     9781 tggacaaagc caacgtgctt gcatcctcgc gtttgtggcg gaagactgtt gaggagacca
     9841 tcaagaacga gttcccgcag ctgacggtgc agcaccagct gatcgactcg gcagccatga
     9901 ttttggtcaa gtcgccaacg aaactcaacg gtgtcattgt caccaacaac atgtttggcg
     9961 acatcatcag tgacgaggcc agtgtgattc ctgggtctct gggcctgctg ccttctgcct
    10021 cgttggcgtc tctgccagac acaaacaagg cgtttggtct ttacgagccc tgccacggct
    10081 cggcgccaga tttgggcccg ggcaaggtca atccattggc cacaattttg tctgccgcca
    10141 tgatgctgaa gctgtcgctg gacttggtgg atgccggccg tgcgatcgag caggccgtca
    10201 agaacgtcct ggatgcaggt atcatgactg ccgatttggg tggaagctcc tcaacacagg
    10261 aagttggtga tgctgttgcg caggaggtgg ccaagctact caagaactaa ataagggaga
    10321 aaaaaaagta ggatcataag ggtaaaatta attcgattac tagtatttat tttctgaaaa
    10381 taaagaatta aaaaaatatt tccttttcat gttcgccagt cacgtccgat atttttccag
    10441 atctctgcga ttgcacgcca tcaaagtgcc tgacaacgcg cctacactcg aagaaaactc
    10501 tgcgtggctc aagaaactgg ccaggtcctc gataagtcca aaaccttcaa gccacacaga
    10561 attgcctaaa tcaacttacg agatacgaca agcttcggag cttgagtccg aaacgaaccc
    10621 agaattcatc aaactaaggc tccctggtgg aaaattcgtc aatgtggtga aaaaagcaga
    10681 tccgcatagc caaccaggaa cctcattttt tacgctttct gtgcccgcat ccgactattt
    10741 cgtggcatcg tcgccgtttt cgagattgcc atccaataaa cgtcccaagg ccgactcttc
    10801 gcagtccttt gcagatctga aggtcgtaaa ggtgagttta aacgccaccg cggtggagct
    10861 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
    10921 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
    10981 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
    11041 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
    11101 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
    11161 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
    11221 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
    11281 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    11341 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    11401 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    11461 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    11521 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    11581 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    11641 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    11701 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
    11761 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    11821 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    11881 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    11941 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    12001 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    12061 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    12121 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    12181 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    12241 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    12301 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    12361 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    12421 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    12481 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    12541 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    12601 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    12661 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    12721 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    12781 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    12841 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    12901 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    12961 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
    13021 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc ttttcatcac
    13081 gtgctataaa aataattata atttaaattt tttaatataa atatataaat taaaaataga
    13141 aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt aaaagactct
    13201 agggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca ggtattaatg
    13261 ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt gattttacat
    13321 tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc taataaatat
    13381 atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt tttttcttca
    13441 ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa aacataaaaa
    13501 taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt
    13561 aagttacagg caagcgatcc gtctaagaaa ccattattat catgacatta acctataaaa
    13621 ataggcgtat cacgaggccc tttcgtc
//
