LOCUS       sequence-329612-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 and tet-inducible GLK1 targeting 
            sgRNA expression for double stranded break formation in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11755)
  TITLE     Burgess Lab Plasmids
REFERENCE   2  (bases 1 to 11755)
  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"
COMMENT     Sequence Label: pNSU299_GLK1
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     370..389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    853..871
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        957..1032
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1045..1107
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             2242..2865
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2315..2334)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     3362..3379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3496..3517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3532..3537,3538..3555,3556..3562)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3570..3586
                     /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     3575..3597
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3594..3610
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3594..3610
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3629..3649
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3631..3649
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3667..4065
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     4069..4085
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4069..4085
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             4097..8200
                     /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             4097..8200
                     /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             4097..8200
                     /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             8213..8233
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8249..8496
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(8265..8283)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(8514..8533)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8515..8533)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8543..8560)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8543..8559)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8552..8574)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8700..9155
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8787..8806)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8997..9018
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(9258..9932)
                     /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(9933..10214)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(10235..10254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10354..10376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10414..10432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10500..10604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10605..10673,10674..11465)
                     /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(10823..10842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(11636..11755,1..469)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
       61 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      121 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      181 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      241 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      301 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      361 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      421 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      481 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcggggga tctgccaatt
      541 gaacataaca tggtagttac atatactagt aatatggttc ggcacacatt aaaagtataa
      601 aaactatctg aattacgaat tacatatatt ggtcataaaa atcaatcaat catcgtgtgt
      661 tttatatgtc tcttatctaa gtataagaat atccatagtt aatattcact tacgctacct
      721 tttaacctgt aatcattgtc aacaggatat gttaacgacc cacattgata aacgctagta
      781 tttctttttc ctcttcttat tggccggctg tctctatact cccctatagt ctgtttcttt
      841 tcgtttcgat tgtccctatc agtgatagag atggcgcaca tggtacgctg tggtgctcgc
      901 ggctgggaac gaaactctgg gagctgcgat tggcaggacc aagggtttcc gcatcggttt
      961 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     1021 ccgagtcggt gcttttttct cgagccatat ccaacttcca atttaatctt tcttttttaa
     1081 ttttcactta tttgcgatac agaaagaaaa aagcgatagt aactattgaa ttttgtttgg
     1141 atttggttag attagatatg gtttctcttt atatttacat gctaaaaatg ggctacacca
     1201 gagatacata attagatata tatacgccag tacaccttat cggcccaagc cttgtcccaa
     1261 ggcagcgttt tgttcttgga aacgctgccc tacacgttcg ctatgcttca agaacttttc
     1321 tgagcacttc atgatgcatg tttgttcctt attggttagc tttgatgttg tgaagtcatt
     1381 gacacagtct gtgaaacatc tttctaccag attagagtac aaacgcatga aatccttcat
     1441 ttgcttttgt tccactactt tttggaactc ttgttgttct ttcctgcagg tagtcgtgca
     1501 atgtatgact ttaagatttg tgagcaggaa gaaaagggag aatcttctaa cgataaaccc
     1561 ttgaaaaact gggtagacta cgctatgttg agttgctacg caggctgcac aattacacga
     1621 gaatgctccc gcctaggatt taaggctaag ggacgtgcaa tgcagacgac agatctaaat
     1681 gaccgtgtcg gtgaagtgtt cgccaaactt ttcggttaac acatgcagtg atgcacgcgc
     1741 gatggtgcta agttacatat atatatatat gtatagccat agtgatgtct aagtaacctt
     1801 tatggtatat ttcttaatgt ggaaagatac tagcgcgcgc acccacacac aagcttcgtc
     1861 ttttcttgaa gaaaagagga agctcgctaa atgggattcc actttccgtt ccctgccagc
     1921 tgatggaaaa aggttagtgg aacgatgaag aataaaaaga gagatccact gaggtgaaat
     1981 ttcagctgac agcgagtttc atgatcgtga tgaacaatgg taacgagttg tggctgttgc
     2041 cagggagggt ggttctcaac ttttaatgta tggccaaatc gctacttggg tttgttatat
     2101 aacaaagaag aaataatgaa ctgattctct tcctccttct tgtcctttct taattctgtt
     2161 gtaattacct tcctttgtaa ttttttttgt aattattctt cttaataatc caaacaaaca
     2221 cacatattac aatatattaa aatgtctaga ttagataaaa gtaaagtgat taacagcgca
     2281 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
     2341 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
     2401 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
     2461 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
     2521 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
     2581 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
     2641 ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
     2701 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
     2761 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
     2821 atatgcggat tagaaaaaca acttaaatgt gaaagtgggt cttaagtctg aagaatgaat
     2881 gatttgatga tttctttttc cctccatttt tcttactgaa tatatcaatg atatagactt
     2941 gtatagttta ttatttcaaa ttaagtagct atatatagtc aagataacgt ttgtttgaca
     3001 cgattacatt attcgtcgac atcttttttc agcctgtcgt ggtagcaatt tgaggagtat
     3061 tattaattga ataggttcat tttgcgctcg cataaacagt tttcgtcagg gacagtatgt
     3121 tggaatgagt ggtaattaat ggtgacatga catgttatag caataacctt gatgtttaca
     3181 tcgtagttta atgtacaccc cgcgaattcg ttcaagtagg agtgcaccaa ttgcaaaggg
     3241 aaaagctgaa tgggcagttc gaaccgcggt ctttcctgcg ttatcccctg attctgtgga
     3301 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3361 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     3421 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3481 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     3541 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     3601 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     3661 ggagctcata gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact
     3721 ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt tcccctcttt
     3781 cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg
     3841 cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct
     3901 tgaaaatttt ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa
     3961 taaacggtct tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt
     4021 ttacttcttg ctcattagaa agaaagcata gcaatctaat ctaagttttc tagaactagt
     4081 ggatcccccg ggaaaaatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag
     4141 cgtcggttgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct
     4201 gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc
     4261 cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag
     4321 aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga
     4381 ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg
     4441 ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat
     4501 atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta
     4561 tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa
     4621 cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct
     4681 tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag
     4741 gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa
     4801 cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa
     4861 cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct
     4921 cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa
     4981 cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc
     5041 tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct
     5101 gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc
     5161 taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt
     5221 tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag
     5281 agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca
     5341 cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga
     5401 taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg taggccccct
     5461 cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc
     5521 ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat
     5581 gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta
     5641 cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag
     5701 aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac
     5761 gaaccggaaa gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt
     5821 cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca
     5881 cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat
     5941 tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg
     6001 cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg
     6061 atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag
     6121 tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca
     6181 gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg
     6241 ccagggggac agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa
     6301 gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa
     6361 gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa
     6421 gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat
     6481 ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta
     6541 cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga
     6601 ctacgacgtg gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa
     6661 agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt
     6721 tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg
     6781 gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg
     6841 cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct
     6901 cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt
     6961 tattactctg aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt
     7021 gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac
     7081 tgcacttatc aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt
     7141 gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa
     7201 gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg
     7261 agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga
     7321 caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt
     7381 taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa
     7441 cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga
     7501 ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa
     7561 aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga
     7621 aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat
     7681 cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc
     7741 tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt
     7801 cttgtatctg gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa
     7861 gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga
     7921 attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa
     7981 taagcacagg gataagccca tcagggagca ggcagaaaac attatccact tgtttactct
     8041 gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg
     8101 gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct
     8161 ctatgaaaca agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa
     8221 gaagaggaag gtgtgatctc ttctcgagtc atgtaattag ttatgtcacg cttacattca
     8281 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     8341 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     8401 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     8461 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat tcgccctata
     8521 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     8581 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     8641 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     8701 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     8761 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     8821 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     8881 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     8941 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     9001 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     9061 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     9121 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     9181 catctgtgcg gtatttcaca ccgcatagat cggcaagtgc acaaacaata cttaaataaa
     9241 tactactcag taataaccta tttcttagca tttttgacga aatttgctat tttgttagag
     9301 tcttttacac catttgtctc cacacctccg cttacatcaa caccaataac gccatttaat
     9361 ctaagcgcat caccaacatt ttctggcgtc agtccaccag ctaacataaa atgtaagctt
     9421 tcggggctct cttgccttcc aacccagtca gaaatcgagt tccaatccaa aagttcacct
     9481 gtcccacctg cttctgaatc aaacaaggga ataaacgaat gaggtttctg tgaagctgca
     9541 ctgagtagta tgttgcagtc ttttggaaat acgagtcttt taataactgg caaaccgagg
     9601 aactcttggt attcttgcca cgactcatct ccatgcagtt ggacgatatc aatgccgtaa
     9661 tcattgacca gagccaaaac atcctcctta ggttgattac gaaacacgcc aaccaagtat
     9721 ttcggagtgc ctgaactatt tttatatgct tttacaagac ttgaaatttt ccttgcaata
     9781 accgggtcaa ttgttctctt tctattgggc acacatataa tacccagcaa gtcagcatcg
     9841 gaatctagag cacattctgc ggcctctgtg ctctgcaagc cgcaaacttt caccaatgga
     9901 ccagaactac ctgtgaaatt aataacagac atactccaag ctgcctttgt gtgcttaatc
     9961 acgtatactc acgtgctcaa tagtcaccaa tgccctccct cttggccctc tccttttctt
    10021 ttttcgaccg aattaattct taatcggcaa aaaaagaaaa gctccggatc aagattgtac
    10081 gtaaggtgac aagctatttt tcaataaaga atatcttcca ctactgccat ctggcgtcat
    10141 aactgcaaag tacacatata ttacgatgct gttctattaa atgcttccta tattatatat
    10201 atagtaatgt cgttgatcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
    10261 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
    10321 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
    10381 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
    10441 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
    10501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
    10561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
    10621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
    10681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
    10741 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
    10801 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
    10861 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
    10921 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
    10981 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
    11041 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
    11101 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
    11161 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
    11221 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
    11281 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
    11341 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
    11401 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
    11461 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
    11521 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
    11581 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
    11641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
    11701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggctt
//
