LOCUS       sequence-365360-       13902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSC101 ori, chl34 resistant. Cas9 induced at 0.4 ug/ml 
            anhydrotetracycline, recombineering proteins induced by a 15 min 
            heat-shock at 42(o)C, I-Scel (induced by 0.1 mM IPTG) is to cleave 
            the pDonor2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13902)
  AUTHORS   Yang DD, Rusch LM, Widney KA, Morgenthaler AB, Copley SD
  TITLE     Synonymous edits in the Escherichia coli genome have substantial and
            condition-dependent effects on fitness.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jan 30;121(5):e2316834121. doi: 
            10.1073/pnas.2316834121. Epub 2024 Jan 22.
  PUBMED    38252823
REFERENCE   2  (bases 1 to 13902)
  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."; date: "2024-01-30"; volume: "121(5):e2316834121. 
            doi"; pages: " 10.1073/pnas.2316834121. Epub 2024 Jan 22"
COMMENT     Sequence Label: pDY118A
FEATURES             Location/Qualifiers
     source          1..13902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(183..863)
                     /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="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     CDS             complement(860..1645)
                     /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"
     protein_bind    2078..2092
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             complement(2384..3043)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2958..2977
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(3156..3869)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     RBS             3958..3966
                     /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)"
     misc_feature    3981..3999
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(4519..5469)
                     /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      5517..5739
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(6295..6921)
                     /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     6826..6845
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(6934..6968)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        6969..7024
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7005..7023
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7060..11163
                     /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             7060..11163
                     /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             11164..11196
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag
                     /note="wild-type LAA variant"
                     /translation="AANDENYALAA"
     terminator      11223..11294
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      11310..11337
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(11365..11382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(12413..12435)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12424..12440
                     /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(12709..13791)
                     /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     13753..13772
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(13792..13869)
                     /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."
ORIGIN
        1 atcgggacca aaacgaaaaa agacgctcga aagcgtctct tttctggaat ttggtaccga
       61 gcatctccgc tgtgctttca gtggatttcg gataacagaa aggccgggaa atacccagcc
      121 tcgctttgta acggagtaga cgaaagtgat tgcgcctacc cggatattat cgtgaggatg
      181 cgtcatcgcc attgctcccc aaatacaaaa ccaatttcag ccagtgcctc gtccattttt
      241 tcgatgaact ccggcacgat ctcgtcaaaa ctcgccatgt acttttcatc ccgctcaatc
      301 acgacataat gcaggccttc acgcttcata cgcgggtcat agttggcaaa gtaccaggca
      361 ttttttcgcg tcacccacat gctgtactgc acctgggcca tgtaagctga ctttatggcc
      421 tcgaaaccac cgagccggaa cttcatgaaa tcccgggagg taaacgggca tttcagttca
      481 aggccgttgc cgtcactgca taaaccatcg ggagagcagg cggtacgcat actttcgtcg
      541 cgatagatga tcggggattc agtaacattc acgccggaag taaattcaaa cagggttctg
      601 gcgtcgttct cgtactgttt tccccaggcc agtgctttag cgttaacttc cggagccaca
      661 ccggtgcaaa cctcagcaag cagggtgtgg aagtaggaca ttttcatgtc aggccacttc
      721 tttccggagc ggggttttgc tatcacgttg tgaacttctg aagcggtgat gacgccgagc
      781 cgtaatttgt gccacgcatc ttccccctgt tcgacagctc tcacatcgat cccggtacgc
      841 tgcaggataa tgtccggtgt catgctgcca ccttctgctc tgcggctttc tgtttcagga
      901 atccaagagc ttttactgct tcggcctgtg tcagttctga cgatgcacga atgtcgcggc
      961 gaaatatctg ggaacagagc ggcaataagt cgtcatccca tgttttatcc agggcgatca
     1021 gcagagtgtt aatctcctgc atggtttcat cgttaaccgg agtgatgtcg cgttccggct
     1081 gacgttctgc agtgtatgca gtattttcga caatgcgctc ggcttcatcc ttgtcataga
     1141 taccagcaaa tccgaaggcc agacgggcac actgaatcat ggctttatga cgtaacatcc
     1201 gtttgggatg cgactgccac ggccccgtga tttctctgcc ttcgcgagtt ttgaatggtt
     1261 cgcggcggca ttcatccatc cattcggtaa cgcagatcgg atgattacgg tccttgcggt
     1321 aaatccggca tgtacaggat tcattgtcct gctcaaagtc catgccatca aactgctggt
     1381 tttcattgat gatgcgggac cagccatcaa cgcccaccac cggaacgatg ccattctgct
     1441 tatcaggaaa ggcgtaaatt tctttcgtcc acggattaag gccgtactgg ttggcaacga
     1501 tcagtaatgc gatgaactgc gcatcgctgg catcaccttt aaatgccgtc tggcgaagag
     1561 tggtgatcag ttcctgtggg tcgacagaat ccatgccgac acgttcagcc agcttcccag
     1621 ccagcgttgc gagtgcagta ctcattcgtt ttatacctct gaatcaatat caacctggtg
     1681 gtgagcaatg gtttcaacca tgtaccggat gtgttctgcc atgcgctcct gaaactcaac
     1741 atcgtcatca aacgcacggg taatggattt tttgctggcc ccgtggcgtt gcaaatgatc
     1801 gatgcatagc gattcaaaca ggtgctgggg caggcctttt tccatgtcgt ctgccagttc
     1861 tgcctctttc tcttcacggg cgagctgctg gtagtgacgc gcccagctct gagcctcaag
     1921 acgatcctga atgtaataag cgttcatggc tgaactcctg aaatagctgt gaaaatatcg
     1981 cccgcgaaat gccgggctga ttagtaatcc ggaatcgcac ttacggccaa tgcttcgttt
     2041 cgtatcacac accccaaagc cttctgcttt gaatgctgcc cttcttcagg gcttaatttt
     2101 taagagcgtc accttcatgg tggtcagtgc gtcctgctga tgtgctcagt atcaccgcca
     2161 gtggtattta tgtcaacacc gccagagata atttatcacc gcagatggtt atctgtatgt
     2221 tttttatatg aatttatttt ttgcaggggg gcattgtttg gtaggtgaga gatctgaatt
     2281 gctatgttta gtgagttgta tctatttatt tttcaataaa tacaattggt tatgtgtttt
     2341 gggggcgatc gtgaggcaaa gaaaacccgg cgctgaggcc gggttacgcc ccgccctgcc
     2401 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa
     2461 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt
     2521 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac
     2581 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag
     2641 ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact
     2701 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga
     2761 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca
     2821 tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa
     2881 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct
     2941 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt
     3001 gggatatatc aacggtggta tatccagtga tttttttttc cataattcaa tccatttact
     3061 atgttatgtt ctgaggggag tgaaaattcc cctaattcga tgaagattct tgctcaattg
     3121 ttatcagcta tgcgccgacc agaacacctt gccgatcagc caaacgtctc ttcaggccac
     3181 tgactagcga taactttccc cacaacggaa caactctcat tgcatgggat cattgggtac
     3241 tgtgggttta gtggttgtaa aaacacctga ccgctatccc tgatcagttt cttgaaggta
     3301 aactcatcac ccccaagtct ggctatgcag aaatcacctg gctcaacagc ctgctcaggg
     3361 tcaacgagaa ttaacattcc gtcaggaaag cttggcttgg agcctgttgg tgcggtcatg
     3421 gaattacctt caacctcaag ccagaatgca gaatcactgg cttttttggt tgtgcttacc
     3481 catctctccg catcaccttt ggtaaaggtt ctaagctcag gtgagaacat ccctgcctga
     3541 acatgagaaa aaacagggta ctcatactca cttctaagtg acggctgcat actaaccgct
     3601 tcatacatct cgtagatttc tctggcgatt gaagggctaa attcttcaac gctaactttg
     3661 agaatttttg taagcaatgc ggcgttataa gcatttaatg cattgatgcc attaaataaa
     3721 gcaccaacgc ctgactgccc catccccatc ttgtctgcga cagattcctg ggataagcca
     3781 agttcatttt tctttttttc ataaattgct ttaaggcgac gtgcgtcctc aagctgctct
     3841 tgtgttaatg gtttcttttt tgtgctcata cgttaaatct atcaccgcaa gggataaatg
     3901 tctaacaccg tgcgtgttga ctattttacc tctggcggtg ataatggttg catgtactaa
     3961 ggaggttgta tggaacaacg agatgtgtat aagagacagc tgacgggttt tgctgcccgc
     4021 aaacgggctg ttctggtgtt gctagtttgt tatcagaatc gcagatccgg cttcagccgg
     4081 tttgccggct gaaagcgcta tttcttccag aattgccatg attttttccc cacgggaggc
     4141 gtcactggct cccgtgttgt cggcagcttt gattcgataa gcagcatcgc ctgtttcagg
     4201 ctgtctatgt gtgactgttg agctgtaaca agttgtctca ggtgttcaat ttcatgttct
     4261 agttgctttg ttttactggt ttcacctgtt ctattaggtg ttacatgctg ttcatctgtt
     4321 acattgtcga tctgttcatg gtgaacagct ttgaatgcac caaaaactcg taaaagctct
     4381 gatgtatcta tcttttttac accgttttca tctgtgcata tggacagttt tccctttgat
     4441 atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc actgatagat
     4501 acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct ctagtgtggt
     4561 tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatacttac tttgcatgtc
     4621 actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag catcgtgtag
     4681 tgtttttctt agtccgttat gtaggtagga atctgatgta atggttgttg gtattttgtc
     4741 accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt gtctatctag
     4801 ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat
     4861 attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt taagcctttt
     4921 aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa ggctaatctc
     4981 tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact cataaatcct
     5041 catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta tgaatttttt
     5101 taactggaaa agataaggca atatctcttc actaaaaact aattctaatt tttcgcttga
     5161 gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag gattcctgat
     5221 ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat aagcattttc
     5281 cctactgatg ttcatcatct gaacgtattg gttataagtg aacgataccg tccgttcttt
     5341 ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa ttagcttggt
     5401 ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga aaacaactaa
     5461 ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat tgagatgggc
     5521 tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta aattctgcta
     5581 gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg ctagaccttt
     5641 gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga aagaataaaa
     5701 aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt cagttccgca
     5761 gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac cttaaaaccc
     5821 taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc cttttgtctc
     5881 cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg
     5941 ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt catgcaagga
     6001 aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt atggcgggtc
     6061 tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct gattttccag
     6121 tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc acccagtaag
     6181 gcagcggtat catcaacagg cttacccgtc ttacttttgt tatcaataaa aaaggccccc
     6241 cgatttggga ggccttattg ttcgtccgtc tcattttcgc cagatatcga cgtcttaaga
     6301 cccactttca catttaagtt gtttttctaa tccgcatatg atcaattcaa ggccgaataa
     6361 gaaggctggc tctgcacctt ggtgatcaaa taattcgata gcttgtcgta ataatggcgg
     6421 catactatca gtagtaggtg tttccctttc ttctttagcg acttgatgct cttgatcttc
     6481 caatacgcaa cctaaagtaa aatgccccac agcgctgagt gcatataatg cattctctag
     6541 tgaaaaacct tgttggcata aaaaggctaa ttgattttcg agagtttcat actgtttttc
     6601 tgtaggccgt gtacctaaat gtacttttgc tccatcgcga tgacttagta aagcacatct
     6661 aaaactttta gcgttattac gtaaaaaatc ttgccagctt tccccttcta aagggcaaaa
     6721 gtgagtatgg tgcctatcta acatctcaat ggctaaggcg tcgagcaaag cccgcttatt
     6781 ttttacatgc caatacaatg taggctgctc tacacctagc ttctgggcga gtttacgggt
     6841 tgttaaacct tcgattccga cctcattaag cagctctaat gcgctgttaa tcactttact
     6901 tttatctaat ctagacatca ttaattccta attgctagca ttgtacctag gactgagcta
     6961 gccataaagt tgacactcta tcgttgatag agttatttta ccactcccta tcagtgatag
     7021 agaaaagaat tcaaaagatc taaagaggac ttcggatcta tggataagaa atactcaata
     7081 ggcttagata tcggcacaaa tagcgtcgga tgggcggtga tcactgatga atataaggtt
     7141 ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc acagtatcaa aaaaaatctt
     7201 ataggggctc ttttatttga cagtggagag acagcggaag cgactcgtct caaacggaca
     7261 gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt atctacagga gattttttca
     7321 aatgagatgg cgaaagtaga tgatagtttc tttcatcgac ttgaagagtc ttttttggtg
     7381 gaagaagaca agaagcatga acgtcatcct atttttggaa atatagtaga tgaagttgct
     7441 tatcatgaga aatatccaac tatctatcat ctgcgaaaaa aattggtaga ttctactgat
     7501 aaagcggatt tgcgcttaat ctatttggcc ttagcgcata tgattaagtt tcgtggtcat
     7561 tttttgattg agggagattt aaatcctgat aatagtgatg tggacaaact atttatccag
     7621 ttggtacaaa cctacaatca attatttgaa gaaaacccta ttaacgcaag tggagtagat
     7681 gctaaagcga ttctttctgc acgattgagt aaatcaagac gattagaaaa tctcattgct
     7741 cagctccccg gtgagaagaa aaatggctta tttgggaatc tcattgcttt gtcattgggt
     7801 ttgaccccta attttaaatc aaattttgat ttggcagaag atgctaaatt acagctttca
     7861 aaagatactt acgatgatga tttagataat ttattggcgc aaattggaga tcaatatgct
     7921 gatttgtttt tggcagctaa gaatttatca gatgctattt tactttcaga tatcctaaga
     7981 gtaaatactg aaataactaa ggctccccta tcagcttcaa tgattaaacg ctacgatgaa
     8041 catcatcaag acttgactct tttaaaagct ttagttcgac aacaacttcc agaaaagtat
     8101 aaagaaatct tttttgatca atcaaaaaac ggatatgcag gttatattga tgggggagct
     8161 agccaagaag aattttataa atttatcaaa ccaattttag aaaaaatgga tggtactgag
     8221 gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca agcaacggac ctttgacaac
     8281 ggctctattc cccatcaaat tcacttgggt gagctgcatg ctattttgag aagacaagaa
     8341 gacttttatc catttttaaa agacaatcgt gagaagattg aaaaaatctt gacttttcga
     8401 attccttatt atgttggtcc attggcgcgt ggcaatagtc gttttgcatg gatgactcgg
     8461 aagtctgaag aaacaattac cccatggaat tttgaagaag ttgtcgataa aggtgcttca
     8521 gctcaatcat ttattgaacg catgacaaac tttgataaaa atcttccaaa tgaaaaagta
     8581 ctaccaaaac atagtttgct ttatgagtat tttacggttt ataacgaatt gacaaaggtc
     8641 aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc
     8701 attgttgatt tactcttcaa aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat
     8761 tatttcaaaa aaatagaatg ttttgatagt gttgaaattt caggagttga agatagattt
     8821 aatgcttcat taggtaccta ccatgatttg ctaaaaatta ttaaagataa agattttttg
     8881 gataatgaag aaaatgaaga tatcttagag gatattgttt taacattgac cttatttgaa
     8941 gatagggaga tgattgagga aagacttaaa acatatgctc acctctttga tgataaggtg
     9001 atgaaacagc ttaaacgtcg ccgttatact ggttggggac gtttgtctcg aaaattgatt
     9061 aatggtatta gggataagca atctggcaaa acaatattag attttttgaa atcagatggt
     9121 tttgccaatc gcaattttat gcagctgatc catgatgata gtttgacatt taaagaagac
     9181 attcaaaaag cacaagtgtc tggacaaggc gatagtttac atgaacatat tgcaaattta
     9241 gctggtagcc ctgctattaa aaaaggtatt ttacagactg taaaagttgt tgatgaattg
     9301 gtcaaagtaa tggggcggca taagccagaa aatatcgtta ttgaaatggc acgtgaaaat
     9361 cagacaactc aaaagggcca gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt
     9421 atcaaagaat taggaagtca gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa
     9481 aatgaaaagc tctatctcta ttatctccaa aatggaagag acatgtatgt ggaccaagaa
     9541 ttagatatta atcgtttaag tgattatgat gtcgatcaca ttgttccaca aagtttcctt
     9601 aaagacgatt caatagacaa taaggtctta acgcgttctg ataaaaatcg tggtaaatcg
     9661 gataacgttc caagtgaaga agtagtcaaa aagatgaaaa actattggag acaacttcta
     9721 aacgccaagt taatcactca acgtaagttt gataatttaa cgaaagctga acgtggaggt
     9781 ttgagtgaac ttgataaagc tggttttatc aaacgccaat tggttgaaac tcgccaaatc
     9841 actaagcatg tggcacaaat tttggatagt cgcatgaata ctaaatacga tgaaaatgat
     9901 aaacttattc gagaggttaa agtgattacc ttaaaatcta aattagtttc tgacttccga
     9961 aaagatttcc aattctataa agtacgtgag attaacaatt accatcatgc ccatgatgcg
    10021 tatctaaatg ccgtcgttgg aactgctttg attaagaaat atccaaaact tgaatcggag
    10081 tttgtctatg gtgattataa agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa
    10141 gaaataggca aagcaaccgc aaaatatttc ttttactcta atatcatgaa cttcttcaaa
    10201 acagaaatta cacttgcaaa tggagagatt cgcaaacgcc ctctaatcga aactaatggg
    10261 gaaactggag aaattgtctg ggataaaggg cgagattttg ccacagtgcg caaagtattg
    10321 tccatgcccc aagtcaatat tgtcaagaaa acagaagtac agacaggcgg attctccaag
    10381 gagtcaattt taccaaaaag aaattcggac aagcttattg ctcgtaaaaa agactgggat
    10441 ccaaaaaaat atggtggttt tgatagtcca acggtagctt attcagtcct agtggttgct
    10501 aaggtggaaa aagggaaatc gaagaagtta aaatccgtta aagagttact agggatcaca
    10561 attatggaaa gaagttcctt tgaaaaaaat ccgattgact ttttagaagc taaaggatat
    10621 aaggaagtta aaaaagactt aatcattaaa ctacctaaat atagtctttt tgagttagaa
    10681 aacggtcgta aacggatgct ggctagtgcc ggagaattac aaaaaggaaa tgagctggct
    10741 ctgccaagca aatatgtgaa ttttttatat ttagctagtc attatgaaaa gttgaagggt
    10801 agtccagaag ataacgaaca aaaacaattg tttgtggagc agcataagca ttatttagat
    10861 gagattattg agcaaatcag tgaattttct aagcgtgtta ttttagcaga tgccaattta
    10921 gataaagttc ttagtgcata taacaaacat agagacaaac caatacgtga acaagcagaa
    10981 aatattattc atttatttac gttgacgaat cttggagctc ccgctgcttt taaatatttt
    11041 gatacaacaa ttgatcgtaa acgatatacg tctacaaaag aagttttaga tgccactctt
    11101 atccatcaat ccatcactgg tctttatgaa acacgcattg atttgagtca gctaggaggt
    11161 gacgctgcta acgacgaaaa ctacgctctg gctgcttaac tcgagtaagg atctccaggc
    11221 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
    11281 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
    11341 atacctaggg atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact
    11401 gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat
    11461 acttaacagg gaagtgagag ggccggacca aaacgaaaaa aggcccccct ttcgggaggc
    11521 ctcttttctg gaatttggta ccgagcggct ggctgaggag ataaataata aacgaggggc
    11581 tgtatgcaca aagcatcttc tgttgagtta agaacgagta tcgagatggc acatagcctt
    11641 gctcaaattg gaatcaggtt tgtgccaata ccagtagaaa cagacgaaga atccatggtt
    11701 attatttcag gaaagtttcg gaggagatag tgttcggcag tttgtacatc atctgcggga
    11761 tcaggtacgg tttgatcagg ttgtagaaga tcaggtaaga catagaatcg atgtagatga
    11821 tcggtttgtt tttgttgatt tttacgtaac agttcagttg gaatttgtta cgcagaccct
    11881 taaccaggta ttctacttct tcgaaagtga aagactgggt gttcagtacg atcgatttgt
    11941 tggtagagtt tttgttgtaa tcccatttac caccatcatc catgaaccag tatgccagag
    12001 acatcggggt caggtagttt tcaaccaggt tgttcgggat ggtttttttg ttgttaacga
    12061 tgaacaggtt agccagtttg ttgaaagctt ggtgtttgaa agtctgggcg ccccaggtga
    12121 ttaccaggtt acccaggtgg ttaacacgtt cttttttgtg cggcggggac agtacccact
    12181 gatcgtacag cagacatacg tggtccatgt atgctttgtt tttccactcg aactgcatac
    12241 agtaggtttt accttcatca cgagaacgga tgtaagcatc acccaggatc agaccgatac
    12301 ctgcttcgaa ctgttcgatg ttcagttcga tcagctggga tttgtattct ttcagcagtt
    12361 tagagttcgg acccaggttc attacctggt ttttttgatg catatgctgt ttcctgtgtg
    12421 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggatgattaa ttgtcaacag
    12481 ctcatttcag aatatttgcc agaaccgtta tgatgtcggc gcaaaaaaca ttatccagaa
    12541 cgggagtgcg ccttgagcga cacgaattat gcagtgattt acgacctgca cagccatacc
    12601 acagcttccg atggctgcct gacgccagaa gcattggtgc accgtgcagt cgatgataag
    12661 ctgtcaaacc agatcaattc gcgctaactt acattaattg cgttgcgctc actgcccgct
    12721 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
    12781 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
    12841 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
    12901 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
    12961 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
    13021 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
    13081 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
    13141 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
    13201 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
    13261 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
    13321 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
    13381 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
    13441 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
    13501 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
    13561 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
    13621 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
    13681 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
    13741 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
    13801 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc
    13861 gatggtgtca acgtaaatgc atgccgctcg ccttccatgg gt
//
