LOCUS       sequence-468702-       12616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 RECKLEEN plasmid, genome editing of Klebsiella pneumoniae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12616)
  AUTHORS   Elsayed EM, Stukenberg D, Meier D, Schmeck B, Becker A
  TITLE     RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform 
            for enhanced genome editing in Klebsiella pneumoniae.
  JOURNAL   Commun Biol. 2025 Oct 30;8(1):1509. doi: 10.1038/s42003-025-08934-8.
  PUBMED    41168338
REFERENCE   2  (bases 1 to 12616)
  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: "Commun 
            Biol."; date: "2025-10-30"; volume: "8(1):1509. doi"; pages: " 
            10.1038/s42003-025-08934-8"
COMMENT     Sequence Label: Reckleen_1_Cas9
FEATURES             Location/Qualifiers
     source          1..12616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             942..953
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(962..996)
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             complement(1684..2766)
                     /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="MKPVTLYDVAEYAGVSYMTVSRVVNQASHVSAKTREKVEAAMVEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIYSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2728..2747
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2843..2848,2849..2864,2865..2871)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2877..2896
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             2927..3343
                     /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             3349..4134
                     /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             4131..4814
                     /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
                     EQWRA"
     terminator      complement(4878..4905)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(4921..4992)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5010..5042)
                     /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"
     CDS             complement(5049..9152)
                     /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(5049..9152)
                     /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"
     protein_bind    9188..9206
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9213..9231
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             9328..9951
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(9401..9420)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        10228..10262
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     protein_bind    10642..10660
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     RBS             10827..10838
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             10845..11558
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      11570..11641
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      11657..11684
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(11930..12518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12010..12029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
       61 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
      121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtc agccaatcga ctggcgagcg
      181 gcatcgcatt cttcgcatcc cgcctctggc ggatgcagga agatcaacgg atctcggccc
      241 agttgaccca gggctgtcgc cacaatgtcg cgggagcgga tcaaccgagc aaaggcatga
      301 ccgactggac cttccttctg aaggctcttc tccttgagcc acctgtccgc caaggcaaag
      361 cgctcacagc agtggtcatt ctcgagataa tcgacgcgta ccaacttgcc atcctgaaga
      421 atggtgcagt gtctcggcac cccataggga acctttgcca tcaactcggc aagatgcagc
      481 gtcgtgttgg catcgtgtcc cacgccgagg agaagtacct gcccatcgag ttcatggaca
      541 cgggcgaccg ggcttgcagg cgagtgaggt ggcaggggca atggatcaga gatgatctgc
      601 tctgcctgtg gccccgctgc cgcaaaggca aatggatggg cgctgcgctt tacatttggc
      661 aggcgccaga atgtgtcaga gacaactcca aggtccggtg taacgggcga cgtggcagga
      721 tcgaacggct cgtcgtccag acctgaccac gagggcatga cgagcgtccc tcccggaccc
      781 agcgcagcac gcagggcctc gatcagtcca agtggcccat cttcgagggg ccggacgcta
      841 cggaaggagc tgtggaccag cagcacaccg ccgggggtaa ccccaaggtt gagaagctga
      901 ccgatgagct cggcttttcg ccattcgtat tgcattgatt atttctcctc tttctctagt
      961 agctagcact atacctagga ctgagctagc cgtaaactcc aacaggacca aaacgaaaaa
     1021 agacgctttt cagcgtctta ttgttcgtct ttggtaccga gctgtgtctg tggctgagag
     1081 agcaggtaca agataaaccc gtctatattc tcaaattctt tcactatcgc acttgtttta
     1141 tgtgaagtcc agatatggac tctggaaccc aactcggagc tctgcgggta gagcgtcaac
     1201 cattcgaacg ttgcgcggtc ttcggagaac cgtaagaggt aatatactcc gatattcatt
     1261 tcagcggtag ctgagtccgt cagttgatac ggtgcgtacc cggagcaaat aggagaggcg
     1321 gttgtagtgg ggggaatgga tagcaagctg cgggtagacc tccaattatt gaaggcctcc
     1381 caaatcgggg ggcctttttt attgataaca aaaggagctc agataaaata tttgctcatg
     1441 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     1501 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca
     1561 tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag cagggattct
     1621 gcaaacccta tgctactccc tcgagccgtc aattgtctga ttcgttacca attaggatcc
     1681 ttatcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1741 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     1801 tgagactggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     1861 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     1921 ataacatgag ctatcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     1981 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     2041 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     2101 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     2161 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     2221 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcctcatg
     2281 ggagtaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     2341 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     2401 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     2461 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     2521 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     2581 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccac
     2641 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     2701 gcgggaaacg gtcatataag agacaccggc atactctgcg acatcgtata acgttactgg
     2761 tttcatattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     2821 ggttttgcgc cattcggctg tgttgacaat taatcatcgg ctcgtataat gtgtggaatt
     2881 gtgagcgctc acaatttact agagtcacac aggaaagtaa tcaatgatgg atattaatac
     2941 tgaaactgag atcaagcaaa agcattcact aacccccttt cctgttttcc taatcagccc
     3001 ggcatttcgc gggcgatatt ttcacagcta tttcaggagt tcagccatga acgcttatta
     3061 cattcaggat cgtcttgagg ctcagagctg ggcgcgtcac taccagcagc tcgcccgtga
     3121 agagaaagag gcagaactgg cagacgacat ggaaaaaggc ctgccccagc acctgtttga
     3181 atcgctatgc atcgatcatt tgcaacgcca cggggccagc aaaaaatcca ttacccgtgc
     3241 gtttgatgac gatgttgagt ttcaggagcg catggcagaa cacatccggt acatggttga
     3301 aaccattgct caccaccagg ttgatattga ttcagaggta taaaacgaat gagtactgca
     3361 ctcgcaacgc tggctgggaa gctggctgaa cgtgtcggca tggattctgt cgacccacag
     3421 gaactgatca ccactcttcg ccagacggca tttaaaggtg atgccagcga tgcgcagttc
     3481 atcgcattac tgatcgttgc caaccagtac ggccttaatc cgtggacgaa agaaatttac
     3541 gcctttcctg ataagcagaa tggcatcgtt ccggtggtgg gcgttgatgg ctggtcccgc
     3601 atcatcaatg aaaaccagca gtttgatggc atggactttg agcaggacaa tgaatcctgt
     3661 acatgccgga tttaccgcaa ggaccgtaat catccgatct gcgttaccga atggatggat
     3721 gaatgccgcc gcgaaccatt caaaactcgc gaaggcagag aaatcacggg gccgtggcag
     3781 tcgcatccca aacggatgtt acgtcataaa gccatgattc agtgtgcccg tctggccttc
     3841 ggatttgctg gtatctatga caaggatgaa gccgagcgca ttgtcgaaaa tactgcatac
     3901 actgcagaac gtcagccgga acgcgacatc actccggtta acgatgaaac catgcaggag
     3961 attaacactc tgctgatcgc cctggataaa acatgggatg acgacttatt gccgctctgt
     4021 tcccagatat ttcgccgcga cattcgtgca tcgtcagaac tgacacaggc cgaagcagta
     4081 aaagctcttg gattcctgaa acagaaagcc gcagagcaga aggtggcagc atgacaccgg
     4141 acattatcct gcagcgtacc gggatcgatg tgagagctgt cgaacagggg gatgatgcgt
     4201 ggcacaaatt acggctcggc gtcatcaccg cttcagaagt tcacaacgtg atagcaaaac
     4261 cccgctccgg aaagaagtgg cctgacatga aaatgtccta cttccacacc ctgcttgctg
     4321 aggtttgcac cggtgtggct ccggaagtta acgctaaagc actggcctgg ggaaaacagt
     4381 acgagaacga cgccagaacc ctgtttgaat tcacttccgg cgtgaatgtt actgaatccc
     4441 cgatcatcta tcgcgacgaa agtatgcgta ccgcctgctc tcccgatggt ttatgcagtg
     4501 acggcaacgg ccttgaactg aaatgcccgt ttacctcccg ggatttcatg aagttccggc
     4561 tcggtggttt cgaggccata aagtcagctt acatggccca ggtgcagtac agcatgtggg
     4621 tgacgcgaaa aaatgcctgg tactttgcca actatgaccc gcgtatgaag cgtgaaggcc
     4681 tgcattatgt cgtgattgag cgggatgaaa agtacatggc gagttttgac gagatcgtgc
     4741 cggagttcat cgaaaaaatg gacgaggcac tggctgaaat tggttttgta tttggggagc
     4801 aatggcgagc ttaagcttaa aaaaaaaaac cccgcccctg acagggcggg gttttttttc
     4861 gcttactagc gtataaacgc agaaaggccc acccgaaggt gagccagtgt gactctagta
     4921 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt
     4981 tcgttttatt tgatgcctgg ttacccctaa gcagccagag cgtagttttc gtcgttagca
     5041 gctaaagcgt cacctcctag ctgactcaaa tcaatgcgtg tttcataaag accagtgatg
     5101 gattgatgga taagagtggc atctaaaact tcttttgtag acgtatatcg tttacgatca
     5161 attgttgtat caaaatattt aaaagcagcg ggagctccaa gattcgtcaa cgtaaataaa
     5221 tgaataatat tttctgcttg ttcacgtatt ggtttgtctc tatgtttgtt atatgcacta
     5281 agaactttat ctaaattggc atctgctaaa ataacacgct tagaaaattc actgatttgc
     5341 tcaataatct catctaaata atgcttatgc tgctccacaa acaattgttt ttgttcgtta
     5401 tcttctggac tacccttcaa cttttcataa tgactagcta aatataaaaa attcacatat
     5461 ttgcttggca gagccagctc atttcctttt tgtaattctc cggcactagc cagcatccgt
     5521 ttacgaccgt tttctaactc aaaaagacta tatttaggta gtttaatgat taagtctttt
     5581 ttaacttcct tatatccttt agcttctaaa aagtcaatcg gatttttttc aaaggaactt
     5641 ctttccataa ttgtgatccc tagtaactct ttaacggatt ttaacttctt cgatttccct
     5701 ttttccacct tagcaaccac taggactgaa taagctaccg ttggactatc aaaaccacca
     5761 tatttttttg gatcccagtc ttttttacga gcaataagct tgtccgaatt tctttttggt
     5821 aaaattgact ccttggagaa tccgcctgtc tgtacttctg ttttcttgac aatattgact
     5881 tggggcatgg acaatacttt gcgcactgtg gcaaaatctc gccctttatc ccagacaatt
     5941 tctccagttt ccccattagt ttcgattaga gggcgtttgc gaatctctcc atttgcaagt
     6001 gtaatttctg ttttgaagaa gttcatgata ttagagtaaa agaaatattt tgcggttgct
     6061 ttgcctattt cttgctcaga cttagcaatc attttacgaa catcataaac tttataatca
     6121 ccatagacaa actccgattc aagttttgga tatttcttaa tcaaagcagt tccaacgacg
     6181 gcatttagat acgcatcatg ggcatgatgg taattgttaa tctcacgtac tttatagaat
     6241 tggaaatctt ttcggaagtc agaaactaat ttagatttta aggtaatcac tttaacctct
     6301 cgaataagtt tatcattttc atcgtattta gtattcatgc gactatccaa aatttgtgcc
     6361 acatgcttag tgatttggcg agtttcaacc aattggcgtt tgataaaacc agctttatca
     6421 agttcactca aacctccacg ttcagctttc gttaaattat caaacttacg ttgagtgatt
     6481 aacttggcgt ttagaagttg tctccaatag tttttcatct ttttgactac ttcttcactt
     6541 ggaacgttat ccgatttacc acgattttta tcagaacgcg ttaagacctt attgtctatt
     6601 gaatcgtctt taaggaaact ttgtggaaca atgtgatcga catcataatc acttaaacga
     6661 ttaatatcta attcttggtc cacatacatg tctcttccat tttggagata atagagatag
     6721 agcttttcat tttgcaattg agtattttca acaggatgct ctttaagaat ctgacttcct
     6781 aattctttga taccttcttc gattcgtttc atacgctctc gcgaattttt ctggcccttt
     6841 tgagttgtct gattttcacg tgccatttca ataacgatat tttctggctt atgccgcccc
     6901 attactttga ccaattcatc aacaactttt acagtctgta aaataccttt tttaatagca
     6961 gggctaccag ctaaatttgc aatatgttca tgtaaactat cgccttgtcc agacacttgt
     7021 gctttttgaa tgtcttcttt aaatgtcaaa ctatcatcat ggatcagctg cataaaattg
     7081 cgattggcaa aaccatctga tttcaaaaaa tctaatattg ttttgccaga ttgcttatcc
     7141 ctaataccat taatcaattt tcgagacaaa cgtccccaac cagtataacg gcgacgttta
     7201 agctgtttca tcaccttatc atcaaagagg tgagcatatg ttttaagtct ttcctcaatc
     7261 atctccctat cttcaaataa ggtcaatgtt aaaacaatat cctctaagat atcttcattt
     7321 tcttcattat ccaaaaaatc tttatcttta ataattttta gcaaatcatg gtaggtacct
     7381 aatgaagcat taaatctatc ttcaactcct gaaatttcaa cactatcaaa acattctatt
     7441 tttttgaaat aatcttcttt taattgctta acggttactt ttcgatttgt tttgaagagt
     7501 aaatcaacaa tggctttctt ctgttcacct gaaagaaatg ctggttttcg cattccttca
     7561 gtaacatatt tgacctttgt caattcgtta taaaccgtaa aatactcata aagcaaacta
     7621 tgttttggta gtactttttc atttggaaga tttttatcaa agtttgtcat gcgttcaata
     7681 aatgattgag ctgaagcacc tttatcgaca acttcttcaa aattccatgg ggtaattgtt
     7741 tcttcagact tccgagtcat ccatgcaaaa cgactattgc cacgcgccaa tggaccaaca
     7801 taataaggaa ttcgaaaagt caagattttt tcaatcttct cacgattgtc ttttaaaaat
     7861 ggataaaagt cttcttgtct tctcaaaata gcatgcagct cacccaagtg aatttgatgg
     7921 ggaatagagc cgttgtcaaa ggtccgttgc ttgcgcagca aatcttcacg atttagtttc
     7981 accaataatt cctcagtacc atccattttt tctaaaattg gtttgataaa tttataaaat
     8041 tcttcttggc tagctccccc atcaatataa cctgcatatc cgttttttga ttgatcaaaa
     8101 aagatttctt tatacttttc tggaagttgt tgtcgaacta aagcttttaa aagagtcaag
     8161 tcttgatgat gttcatcgta gcgtttaatc attgaagctg ataggggagc cttagttatt
     8221 tcagtattta ctcttaggat atctgaaagt aaaatagcat ctgataaatt cttagctgcc
     8281 aaaaacaaat cagcatattg atctccaatt tgcgccaata aattatctaa atcatcatcg
     8341 taagtatctt ttgaaagctg taatttagca tcttctgcca aatcaaaatt tgatttaaaa
     8401 ttaggggtca aacccaatga caaagcaatg agattcccaa ataagccatt tttcttctca
     8461 ccggggagct gagcaatgag attttctaat cgtcttgatt tactcaatcg tgcagaaaga
     8521 atcgctttag catctactcc acttgcgtta atagggtttt cttcaaataa ttgattgtag
     8581 gtttgtacca actggataaa tagtttgtcc acatcactat tatcaggatt taaatctccc
     8641 tcaatcaaaa aatgaccacg aaacttaatc atatgcgcta aggccaaata gattaagcgc
     8701 aaatccgctt tatcagtaga atctaccaat ttttttcgca gatgatagat agttggatat
     8761 ttctcatgat aagcaacttc atctactata tttccaaaaa taggatgacg ttcatgcttc
     8821 ttgtcttctt ccaccaaaaa agactcttca agtcgatgaa agaaactatc atctactttc
     8881 gccatctcat ttgaaaaaat ctcctgtaga taacaaatac gattcttccg acgtgtatac
     8941 cttctacgag ctgtccgttt cagacgagtc gcttccgctg tctctccact gtcaaataaa
     9001 agagccccta taagattttt tttgatactg tggcggtctg tatttcccag aaccttgaac
     9061 tttttagacg gaaccttata ttcatcagtg atcaccgccc atccgacgct atttgtgccg
     9121 atatctaagc ctattgagta tttcttatcc attgattagt cctgtgtgac tctagtagtg
     9181 ctcagtatct ctatcactga tagggatgtc aatctctatc actgataggg aggtcagtgc
     9241 gtcctgctga aaacagccga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     9301 gaagagagtc aattcagggt ggtgaatatg tccagattag ataaaagtaa agtgattaac
     9361 agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc
     9421 cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg
     9481 ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa
     9541 ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta
     9601 agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa
     9661 actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta
     9721 tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat
     9781 caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga
     9841 caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa
     9901 ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtcctg ataaggatcc
     9961 taattggtaa cgaatcagaa ttgacggctc gagggagtag catagggttt gcagaatccc
    10021 tgcttcgtcc atttgacagg cacattatgc atcgatgata agctgtcaaa catgagcaga
    10081 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
    10141 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagca
    10201 aatattttat ctgagctcca atgggagttg acagctagct cagtcctagg gattgtgcta
    10261 gctactagag tcacacagga aagtaatcaa tgaacattaa cgacctcata cgagagatta
    10321 agaacaaaga ttacaccgtc aaactgtcag gaactgatag taactcaatc acccagctta
    10381 ttatcagggt aaacaatgat gggaatgaat atgtgatatc tgagagcgaa aacgagtcta
    10441 tcgtcgagaa attcatttcc gcttttaaga acgggtggaa tcaggaatat gaggatgaag
    10501 aagaatttta caatgacatg cagacgatca cgttgaaaag tgaactgaac taagcttaac
    10561 gccaaaaacc ccgcttcggc ggggtttttc cgccgctgct tcaaatcact gtaatgatct
    10621 taattcctaa tttttgttga cactctatca ttgatagagt tattttagtc cagagaccga
    10681 aagtgaaacg tgatttcatg cgtcattttg aacattttgt aaatcttatt taataatgtg
    10741 tgcggcaatt cacatttaat ttatgaatgt tttcttaaca tcgcggcaac tcaagaaacg
    10801 gcaggttcgg atcttagcta ctagagaaag aggagaaata ctagatgcgt aaaggcgaag
    10861 aacttttcac tggcgtagta ccaattttag tggagctgga cggagatgta aatggtcata
    10921 agttttcagt tcgaggagaa ggcgaaggag atgcaactaa cggtaagctg acactaaagt
    10981 ttatctgtac cacgggtaaa ctgccggtcc catggccgac actggttact acacttactt
    11041 atggtgtaca atgctttgct cgttatcctg accatatgaa gcagcacgat ttcttcaaat
    11101 cggccatgcc tgaaggatac gtgcaagaac gtacaattag ctttaaagat gacggcacct
    11161 ataaaacgcg cgccgaagtt aaattcgagg gtgacacatt ggttaatcgt atagaactta
    11221 agggcattga cttcaaagaa gatggcaaca tccttggcca taaactggaa tataatttta
    11281 actctcacaa tgtctacatt acggcggata aacaaaagaa tggcattaaa gcgaatttca
    11341 aaatccgcca caatgtcgaa gacggctcgg ttcaactggc ggaccattat cagcagaaca
    11401 ccccaatcgg tgatggcccg gttctgttac ctgataatca ttacctttct actcaaagcg
    11461 ttttatctaa agatcctaac gagaagcgtg atcatatggt tctactggaa tttgttaccg
    11521 cagctggtat cacgcacggc atggatgagc tgtataaata accaggcatc aaataaaacg
    11581 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
    11641 ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata ggtctcagtt
    11701 tcagagctat gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg
    11761 aaaaagtggc accgagtcgg tgcttttttc tcggtaccaa attccagaaa agaggcctcc
    11821 cgaaaggggg gccttttttc gttttggtcc accctgcacg ataacactga ggtagctgcg
    11881 attagacagc cagcagctga accgtaaaaa aggccgcgtt gctggcgttt ttccacaggc
    11941 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
    12001 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
    12061 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
    12121 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
    12181 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
    12241 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
    12301 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
    12361 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
    12421 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
    12481 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
    12541 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
    12601 caaaaaggat cttcac
//
