LOCUS       sequence-254797-       12042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of cas9 and anhydrotetracycline/tetracycline
            inducible expression of lamda RED. Useful variant of Plasmid #62225 
            when arabinose induction is not possible. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12042)
  AUTHORS   Johnston CD, Cotton SL, Rittling SR, Starr JR, Borisy GG, Dewhirst 
            FE, Lemon KP
  TITLE     Systematic evasion of the restriction-modification barrier in 
            bacteria.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 May 16. pii: 1820256116. doi: 
            10.1073/pnas.1820256116.
  PUBMED    31097593
REFERENCE   2  (bases 1 to 12042)
  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: "Proc Natl 
            Acad Sci U S A. 2019 May 16. pii: 1820256116. doi: 
            10.1073/pnas.1820256116."
COMMENT     Sequence Label: pCasTet-lambda
FEATURES             Location/Qualifiers
     source          1..12042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(14..89)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    119..135
                     /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)."
     CDS             complement(404..1486)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1448..1467
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1487..1564)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             complement(1697..2647)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2695..2917
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(3569..4384)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3569..4384)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4292..4311
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4376..4395
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_RNA        complement(4540..4618)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             4914..9020
                     /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             4914..9017
                     /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(9065..9691)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     9596..9615
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        join(9757..9775,9776..9781,9782..9798,9799..9804,
                     9805..9830)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    9757..9775
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9782..9800
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             9869..9877
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             9883..10299
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     CDS             10305..11090
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             11087..11767
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     terminator      11768..12012
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
ORIGIN
        1 gatctcaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
       61 ttttaacttg ctatttctag ctctaaaacc tggtaacagg attagcagat gtgtgaaatt
      121 gttatccgct cacaattcca cacaacatac gagccggatg attaattgtc aacagctcat
      181 ttcagaatat ttgccagaac cgttatgatg tcggcgcaaa aaacattatc cagaacggga
      241 gtgcgccttg agcgacacga attatgcagt gatttacgac ctgcacagcc ataccacagc
      301 ttccgatggc tgcctgacgc cagaagcatt ggtgcaccgt gcagtcgatg ataagctgtc
      361 aaaccagatc aattcgcgct aacttacatt aattgcgttg cgctcactgc ccgctttcca
      421 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      481 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
      541 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
      601 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
      661 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
      721 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
      781 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      841 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      901 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      961 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     1021 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     1081 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1141 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1201 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     1261 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     1321 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     1381 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     1441 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1501 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg
     1561 tgtcaacgta aatgcatgcc gctcgccttc catgggtatg gacagttttc cctttgatat
     1621 gtaacggtga acagttgttc tacttttgtt tgttagtctt gatgcttcac tgatagatac
     1681 aagagccata agaacctcag atccttccgt atttagccag tatgttctct agtgtggttc
     1741 gttgtttttg cgtgagccat gagaacgaac cattgagatc atacttactt tgcatgtcac
     1801 tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc agttaaagca tcgtgtagtg
     1861 tttttcttag tccgttatgt aggtaggaat ctgatgtaat ggttgttggt attttgtcac
     1921 cattcatttt tatctggttg ttctcaagtt cggttacgag atccatttgt ctatctagtt
     1981 caacttggaa aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc aatttcatat
     2041 tgctgtaagt gtttaaatct ttacttattg gtttcaaaac ccattggtta agccttttaa
     2101 actcatggta gttattttca agcattaaca tgaacttaaa ttcatcaagg ctaatctcta
     2161 tatttgcctt gtgagttttc ttttgtgtta gttcttttaa taaccactca taaatcctca
     2221 tagagtattt gttttcaaaa gacttaacat gttccagatt atattttatg aattttttta
     2281 actggaaaag ataaggcaat atctcttcac taaaaactaa ttctaatttt tcgcttgaga
     2341 acttggcata gtttgtccac tggaaaatct caaagccttt aaccaaagga ttcctgattt
     2401 ccacagttct cgtcatcagc tctctggttg ctttagctaa tacaccataa gcattttccc
     2461 tactgatgtt catcatctga acgtattggt tataagtgaa cgataccgtc cgttctttcc
     2521 ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt agcttggttt
     2581 catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa acaactaatt
     2641 cagacataca tctcaattgg tctaggtgat tttaatcact ataccaattg agatgggcta
     2701 gtcaatgata attactagtc cttttccttt gagttgtggg tatctgtaaa ttctgctaga
     2761 cctttgctgg aaaacttgta aattctgcta gaccctctgt aaattccgct agacctttgt
     2821 gtgttttttt tgtttatatt caagtggtta taatttatag aataaagaaa gaataaaaaa
     2881 agataaaaag aatagatccc agccctgtgt ataactcact actttagtca gttccgcagt
     2941 attacaaaag gatgtcgcaa acgctgtttg ctcctctaca aaacagacct taaaacccta
     3001 aaggcttaag tagcaccctc gcaagctcgg ttgcggccgc aatcgggcaa atcgctgaat
     3061 attccttttg tctccgacca tcaggcacct gagtcgctgt ctttttcgtg acattcagtt
     3121 cgctgcgctc acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg
     3181 gattcatgca aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg
     3241 ttttatggcg ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc
     3301 ctctgatttt ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta
     3361 atgcacccag taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact
     3421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3541 acgtttccaa ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact
     3601 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3661 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3721 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3781 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca
     3841 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3901 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3961 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     4021 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     4081 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     4141 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     4201 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     4261 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     4321 cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt tgaatatggc
     4381 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata
     4441 tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttccctgc
     4501 agggtttgca gtcagagtag aatagaagta tcaaaaaaag caccgactcg gtgccacttt
     4561 ttcaagttga taacggacta gccttatttt aacttgctat gctgttttga atggttccaa
     4621 caagattatt ttataacttt tataacaaat aatcaaggag aaattcaaag aaatttatca
     4681 gccataaaac aatacttaat actatagaat gataacaaaa taaactactt tttaaaagaa
     4741 ttttgtgtta taatctattt attattaagt attgggtaat attttttgaa gagatatttt
     4801 gaaaaagaaa aattaaagca tattaaacta atttcggagg tcattaaaac tattattgaa
     4861 atcatcaaac tcattatgga tttaatttaa actttttatt ttaggaggca aaaatggata
     4921 agaaatactc aataggctta gatatcggca caaatagcgt cggatgggcg gtgatcactg
     4981 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
     5041 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
     5101 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
     5161 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
     5221 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
     5281 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
     5341 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
     5401 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
     5461 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
     5521 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
     5581 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     5641 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     5701 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     5761 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     5821 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     5881 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     5941 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     6001 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     6061 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     6121 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt
     6181 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     6241 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     6301 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg
     6361 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     6421 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     6481 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     6541 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     6601 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     6661 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag
     6721 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     6781 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     6841 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     6901 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     6961 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     7021 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     7081 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     7141 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     7201 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac
     7261 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa
     7321 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt
     7381 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat cacattgttc
     7441 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa
     7501 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt
     7561 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag
     7621 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg
     7681 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat
     7741 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag
     7801 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc
     7861 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa
     7921 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg
     7981 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca
     8041 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa
     8101 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag
     8161 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag
     8221 gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta
     8281 aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag
     8341 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt
     8401 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag
     8461 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc
     8521 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag
     8581 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg
     8641 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata
     8701 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag
     8761 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac
     8821 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg
     8881 cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt
     8941 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga
     9001 gtcagctagg aggtgactga agtatatttt agatgaagat tatttcttaa tctagacatg
     9061 agcgttaaga cccactttca catttaagtt gtttttctaa tccgcatatg atcaattcaa
     9121 ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa taattcgata gcttgtcgta
     9181 ataatggcgg catactatca gtagtaggtg tttccctttc ttctttagcg acttgatgct
     9241 cttgatcttc caatacgcaa cctaaagtaa aatgccccac agcgctgagt gcatataatg
     9301 cattctctag tgaaaaacct tgttggcata aaaaggctaa ttgattttcg agagtttcat
     9361 actgtttttc tgtaggccgt gtacctaaat gtacttttgc tccatcgcga tgacttagta
     9421 aagcacatct aaaactttta gcgttattac gtaaaaaatc ttgccagctt tccccttcta
     9481 aagggcaaaa gtgagtatgg tgcctatcta acatctcaat ggctaaggcg tcgagcaaag
     9541 cccgcttatt ttttacatgc caatacaatg taggctgctc tacacctagc ttctgggcga
     9601 gtttacgggt tgttaaacct tcgattccga cctcattaag cagctctaat gcgctgttaa
     9661 tcactttact tttatctaat ctagacatca ttaattccta atttttgggt ctactctatc
     9721 attgatagcg tgcctttacc actccctatc atcgtttccc tatcagtgat agagattgac
     9781 atccctatca gtgatagaga tactgagcac atcagcagga cgcactgacc ggcggccgcg
     9841 cgaattcgag ctcggtaccc gggtgaccta aggaggtaaa taatggatat taatactgaa
     9901 actgagatca agcaaaagca ttcactaacc ccctttcctg ttttcctaat cagcccggca
     9961 tttcgcgggc gatattttca cagctatttc aggagttcag ccatgaacgc ttattacatt
    10021 caggatcgtc ttgaggctca gagctgggcg cgtcactacc agcagctcgc ccgtgaagag
    10081 aaagaggcag aactggcaga cgacatggaa aaaggcctgc cccagcacct gtttgaatcg
    10141 ctatgcatcg atcatttgca acgccacggg gccagcaaaa aatccattac ccgtgcgttt
    10201 gatgacgatg ttgagtttca ggagcgcatg gcagaacaca tccggtacat ggttgaaacc
    10261 attgctcacc accaggttga tattgattca gaggtataaa acgaatgagt actgcactcg
    10321 caacgctggc tgggaagctg gctgaacgtg tcggcatgga ttctgtcgac ccacaggaac
    10381 tgatcaccac tcttcgccag acggcattta aaggtgatgc cagcgatgcg cagttcatcg
    10441 cattactgat cgttgccaac cagtacggcc ttaatccgtg gacgaaagaa atttacgcct
    10501 ttcctgataa gcagaatggc atcgttccgg tggtgggcgt tgatggctgg tcccgcatca
    10561 tcaatgaaaa ccagcagttt gatggcatgg actttgagca ggacaatgaa tcctgtacat
    10621 gccggattta ccgcaaggac cgtaatcatc cgatctgcgt taccgaatgg atggatgaat
    10681 gccgccgcga accattcaaa actcgcgaag gcagagaaat cacggggccg tggcagtcgc
    10741 atcccaaacg gatgttacgt cataaagcca tgattcagtg tgcccgtctg gccttcggat
    10801 ttgctggtat ctatgacaag gatgaagccg agcgcattgt cgaaaatact gcatacactg
    10861 cagaacgtca gccggaacgc gacatcactc cggttaacga tgaaaccatg caggagatta
    10921 acactctgct gatcgccctg gataaaacat gggatgacga cttattgccg ctctgttccc
    10981 agatatttcg ccgcgacatt cgtgcatcgt cagaactgac acaggccgaa gcagtaaaag
    11041 ctcttggatt cctgaaacag aaagccgcag agcagaaggt ggcagcatga caccggacat
    11101 tatcctgcag cgtaccggga tcgatgtgag agctgtcgaa cagggggatg atgcgtggca
    11161 caaattacgg ctcggcgtca tcaccgcttc agaagttcac aacgtgatag caaaaccccg
    11221 ctccggaaag aagtggcctg acatgaaaat gtcctacttc cacaccctgc ttgctgaggt
    11281 ttgcaccggt gtggctccgg aagttaacgc taaagcactg gcctggggaa aacagtacga
    11341 gaacgacgcc agaaccctgt ttgaattcac ttccggcgtg aatgttactg aatccccgat
    11401 catctatcgc gacgaaagta tgcgtaccgc ctgctctccc gatggtttat gcagtgacgg
    11461 caacggcctt gaactgaaat gcccgtttac ctcccgggat ttcatgaagt tccggctcgg
    11521 tggtttcgag gccataaagt cagcttacat ggcccaggtg cagtacagca tgtgggtgac
    11581 gcgaaaaaat gcctggtact ttgccaacta tgacccgcgt atgaagcgtg aaggcctgca
    11641 ttatgtcgtg attgagcggg atgaaaagta catggcgagt tttgacgaga tcgtgccgga
    11701 gttcatcgaa aaaatggacg aggcactggc tgaaattggt tttgtatttg gggagcaatg
    11761 gcgatgacgc atcctcacga taatatccgg gtaggcgcaa tcactttcgt ctactccgtt
    11821 acaaagcgag gctgggtatt tcccggcctt tctgttatcc gaaatccact gaaagcacag
    11881 cggctggctg aggagataaa taataaacga ggggctgtat gcacaaagca tcttctgttg
    11941 agttaagaac gagtatcgag atggcacata gccttgctca aattggaatc aggtttgtgc
    12001 caataccagt agaaacagac gaagaatcca tgggtatgga ca
//
