LOCUS       sequence-462306-       11247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast CEN plasmid for galactose-inducible expression of spCas9 and 
            Ec86 reverse transcriptase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11247)
  AUTHORS   Stuecker TN, Hood SE, Molina Pineda J, Lenaduwe S, Winter J, Sadhu 
            MJ, Lewis JA
  TITLE     Improved vectors for retron-mediated CRISPR-Cas9 genome editing in 
            Saccharomyces cerevisiae.
  JOURNAL   G3 (Bethesda). 2025 Aug 4:jkaf175. doi: 10.1093/g3journal/jkaf175.
  PUBMED    40758833
REFERENCE   2  (bases 1 to 11247)
  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: "G3 
            (Bethesda). 2025 Aug 4:jkaf175. doi: 10.1093/g3journal/jkaf175."
COMMENT     Sequence Label: pCas9-EcRT-GAL-Nat
FEATURES             Location/Qualifiers
     source          1..11247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(130..198,199..990)
                     /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(348..367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1161..1749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1650..1669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1903..1920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2037..2058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2073..2078,2079..2096,2097..2103)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2111..2127
                     /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     2116..2138
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2135..2151
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2135..2151
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2172..2190
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     gene            2318..3437
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2318..2661
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2662..3234
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNALAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      3240..3437
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3553..3572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3554..3572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3579..3596)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3579..3595)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3588..3610)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3736..4191
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3823..3842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4033..4054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5496..5516)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        5738..6179
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    5738..5855
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     6150..6173
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             6212..10315
                     /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             6212..10315
                     /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             6212..10315
                     /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             10328..10348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(10380..10398)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      10410..10599
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     10613..10635
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10673..10691)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    10732..11235
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
       61 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
      121 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
      181 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
      241 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
      301 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
      361 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
      421 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
      481 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
      541 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
      601 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
      661 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
      721 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
      781 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
      841 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
      901 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
      961 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     1021 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1081 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1141 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1201 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1261 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     1321 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     1381 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     1441 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     1501 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     1561 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     1621 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     1681 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     1741 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     1801 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     1861 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     1921 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     1981 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2041 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     2101 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     2161 gccaagctcg aaattaaccc tcactaaagg gaacaaaagc tgctagagcg gccgccagct
     2221 gaagcttcgt acgctgcagg tcgacggatc cccgggttaa ttaaggcgcg ccagatctgt
     2281 ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac atggaggccc agaataccct
     2341 ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt gactgtcgcc cgtacattta
     2401 gcccatacat ccccatgtat aatcatttgc atccatacat tttgatggcc gcacggcgcg
     2461 aagcaaaaat tacggctcct cgctgcagac ctgcgagcag ggaaacgctc ccctcacaga
     2521 cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat ataaaaggtt aggatttgcc
     2581 actgaggttc ttctttcata tacttccttt taaaatcttg ctaggataca gttctcacat
     2641 cacatccgaa cataaacaac catgggtacc actcttgacg acacggctta ccggtaccgc
     2701 accagtgtcc cgggggacgc cgaggccatc gaggcactgg atgggtcctt caccaccgac
     2761 accgtcttcc gcgtcaccgc caccggggac ggcttcaccc tgcgggaggt gccggtggac
     2821 ccgcccctga ccaaggtgtt ccccgacgac gaatcggacg acgaatcgga cgacggggag
     2881 gacggcgacc cggactcccg gacgttcgtc gcgtacgggg acgacggcga cctggcgggc
     2941 ttcgtggtcg tctcgtactc cggctggaac cgccggctga ccgtcgagga catcgaggtc
     3001 gccccggagc accgggggca cggggtcggg cgcgcgttga tggggctcgc gacggagttc
     3061 gcccgcgagc ggggcgccgg gcacctctgg ctggaggtca ccaacgtcaa cgcactggcg
     3121 atccacgcgt accggcggat ggggttcacc ctctgcggcc tggacaccgc cctgtacgac
     3181 ggcaccgcct cggacggcga gcaggcgctc tacatgagca tgccctgccc ctaatcagta
     3241 ctgacaataa aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg
     3301 tagttgttct attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca
     3361 tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa
     3421 tgctggtcgc tatactgctg tcgattcgat actaacgccg ccatccagtg tcgaaaacga
     3481 gctcgaattc atcgatgata tcagatccac tagtggccta tgcggccgcc accgcggtgg
     3541 agctccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt
     3601 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3661 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3721 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3781 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3841 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3901 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3961 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4021 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4081 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4141 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     4201 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagggtaat aactgatata
     4261 attaaattga agctctaatt tgtgagttta gtatacatgc atgcgaattg catatctttc
     4321 catagatttt caatttttga tgtctccatg gtggtacaga gaacttgtaa acaattcggt
     4381 ccctacatgt gaggaaattc gctgtgacac ttttatcact gaactccaaa tttaaaaaat
     4441 agcataaaat tcgttataca gcaaatctat gtgttgcaat taagaactaa aagatataga
     4501 gtgcatattt tcaagaagga tagtaagctg gcaaatcaag tcttagcctt gttcaatggg
     4561 ttcttaccgt acttcttttc caacttagag atgtaggtaa tcaatcttct gtgggactta
     4621 gaatcaacgg acaagatgaa agacaaccaa cctctaacat gttcgatttc ggaggattta
     4681 ccgcagaaaa tatggtggat cttggctctg atttctttgt acttttctct accaatacca
     4741 accttttctt gggagataac caaaccggta acttttcttt gggatcttgg accagaaata
     4801 caggtctttt tggagttgat gaccaaacct tctgatggaa taatggagaa caagaagtct
     4861 ctggccttaa caactttctt catagattga gcagacaagg tcaagtcatc agcgtatcta
     4921 gtgtatatca aacctctaga accagcataa ccttgaattc tgtagtccaa cttggagcaa
     4981 atcaagttag ccaattttgg agaagatgga gcaccttgtg gcaacaagtt cttgtagcag
     5041 caaatcttgg tcaaaacaga ggagatcaat ctgttgtaac ccaaagaatg gaaaacaccg
     5101 aaaaccttgt tagcagtcaa agatgggaag aaatcttcca agtcgatgtt caaaatgaag
     5161 ttggcaccaa tatgtggagt ggcattgttc aagatggatt ggtgcttttc aaaaccgata
     5221 gaaaatgggg aggaggataa cttgtccaag atgtttctca aaacccaacc ttgtaaagcc
     5281 ttcaattctc tggatggttg gtagatagtt ctcattctct tttctggacc cttcttttca
     5341 acagtgtaga ttctgtatct gaaatcggcg gtgtagatca ataatctcaa ggtttcaaca
     5401 gaaattctgg tagccttaga catatcgtgc aagttgttca tgactggcaa acccaaattt
     5461 ctcaatctga aggtgttcaa gtattcagcg gacttgacct ttcttttctt ctttggtggc
     5521 atttatattg aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa
     5581 taatcatatt acatggcatt accaccatat acatatccat atctaatctt acttatatgt
     5641 tgtggaaatg taaagagccc cattatctta gcctaaaaaa accttctctt tggaactttc
     5701 agtaatacgc ttaactgctc attgctatat tgaagtacgg attagaagcc gccgagcggg
     5761 cgacagccct ccgacggaag actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc
     5821 tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa taaagattct acaatactag
     5881 cttttatggt tatgaagagg aaaaattggc agtaacctgg ccccacaaac cttcaaatta
     5941 acgaatcaaa ttaacaacca taggatgata atgcgattag ttttttagcc ttatttctgg
     6001 ggtaattaat cagcgaagcg atgatttttg atctattaac agatatataa atggaaaagc
     6061 tgcataacca ctttaactaa tactttcaac attttcagtt tgtattactt cttattcaaa
     6121 tgtcataaaa gtatcaacaa aaaattgtta atatacctct atactttaac gtcaaggaga
     6181 aaaaacaact agtggatccc ccgggaaaaa aatggacaag aagtactcca ttgggctcga
     6241 tatcggcaca aacagcgtcg gttgggccgt cattacggac gagtacaagg tgccgagcaa
     6301 aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggcgc
     6361 cctcctgttc gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg
     6421 cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat
     6481 ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga
     6541 taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga
     6601 aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg ataaggctga
     6661 cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac acttcctcat
     6721 cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca
     6781 gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg acgccaaagc
     6841 aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc
     6901 tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc
     6961 caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga gcaaagacac
     7021 ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt
     7081 tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac
     7141 ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca
     7201 agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat
     7261 tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga
     7321 ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct
     7381 ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat
     7441 cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta
     7501 cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta
     7561 ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcaga
     7621 agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc
     7681 cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa
     7741 acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt
     7801 cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga
     7861 cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa
     7921 aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc
     7981 cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga
     8041 ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga
     8101 gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca
     8161 gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga tcaatgggat
     8221 ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa
     8281 ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa
     8341 agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag
     8401 cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt
     8461 aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac
     8521 ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga
     8581 actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa
     8641 gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat
     8701 caatcggctc tccgactacg acgtggatca tatcgtgccc cagtcttttc tcaaagatga
     8761 ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt
     8821 cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa
     8881 actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga
     8941 gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca
     9001 cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat
     9061 tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt
     9121 tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa
     9181 tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg aatttgttta
     9241 cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg
     9301 caaggccacc gctaagtact tcttttacag caatattatg aattttttca agaccgagat
     9361 tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg
     9421 agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc
     9481 gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat
     9541 cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa
     9601 atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga
     9661 gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga
     9721 gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt
     9781 caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccg
     9841 gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc
     9901 taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga
     9961 agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat
    10021 cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt
    10081 gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat
    10141 ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac
    10201 catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca
    10261 gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg gagacagcag
    10321 ggctgacccc aagaagaaga ggaaggtgtg atctcttctc gagtcatgta attagttatg
    10381 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
    10441 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
    10501 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
    10561 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttc atccgggagc
    10621 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
    10681 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac ggatcgcttg
    10741 cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg
    10801 tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag
    10861 agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta
    10921 ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac
    10981 gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga
    11041 aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg
    11101 cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct
    11161 ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat tataattatt
    11221 tttatagcac gtgatgaaaa ggaccca
//
