LOCUS       sequence-430405-       14685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To create delta(glmS,nadE) working strains and maintaining their 
            viability.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14685)
  AUTHORS   Amrofell MB, Rengarajan S, Vo ST, Ramirez Tovar ES, LoBello L, 
            Dantas G, Moon TS
  TITLE     Engineering E. coli strains using antibiotic-resistance-gene-free 
            plasmids.
  JOURNAL   Cell Rep Methods. 2023 Dec 18;3(12):100669. doi: 
            10.1016/j.crmeth.2023.100669. Epub 2023 Dec 11.
  PUBMED    38086386
REFERENCE   2  (bases 1 to 14685)
  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: "Cell Rep 
            Methods."; date: "2023-12-18"; volume: "3(12):100669. doi"; pages: "
            10.1016/j.crmeth.2023.100669. Epub 2023 Dec 11"
COMMENT     Sequence Label: pMBA323
FEATURES             Location/Qualifiers
     source          1..14685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        52..86
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     promoter        1124..1158
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             join(1236..1322,1323..2657)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     terminator      complement(2712..2956)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(2957..3637)
                     /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"
     CDS             complement(3634..4419)
                     /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             complement(4425..4841)
                     /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"
     RBS             4850..4858
                     /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)"
     promoter        complement(4885..5169)
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     complement(4943..4962)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             5196..6074
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     terminator      complement(6125..6156)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(6290..6913)
                     /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     6821..6840
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(6914..6991)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    7048..7066
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7073..7091
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        7122..7197
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             7218..7247
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             7263..7280
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7336..7353)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      7506..7552
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    7602..7623
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        7624..7658
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     CDS             7694..11797
                     /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             7694..11797
                     /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             11807..11830
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        11878..11981
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11982..12050,12051..12842)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(12200..12219)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13464..13686
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             13734..14684
                     /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"
ORIGIN
        1 gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa attgacagct
       61 agctcagtcc taggtataat gctagcgaac gatacctaat agaaaatagg aattgacacc
      121 aatgacattg caacaacaaa taataaaggc gctgggcgca aaaccgcaga ttaatgctga
      181 agaggaaatt cgtcgtagta tcgattttct gaaaagctac ctgcaaactt atccgttcat
      241 taaatcactg gtgctcggga tcagcggcgg tcaggactcc acgcttgccg gtaagctgtg
      301 ccagatggcg attaatgagc tgcgccagga aaccggcaac gaatcactgc aatttattgc
      361 cgtacgcctg ccctatggtg tccaggccga cgaacaagat tgccaggatg ccatcgcctt
      421 tattcaaccg gatcgcgtat taaccgttaa tatcaagggc gcggtactcg ccagcgagca
      481 ggcattgcgg gaagcaggca ttgaactgag cgattttgtc cgtggcaatg aaaaagcgcg
      541 tgagcggatg aaagcacaat atagcattgc gggtatgacc agcggtgtcg tggtgggcac
      601 cgatcatgct gcagaagcta ttaccggatt cttcactaaa tatggtgacg gcggtacgga
      661 cattaatccg ctgtatcgtc tcaacaaacg tcagggtaaa cagttactgg cggcattagg
      721 ttgcccggaa cacctttata agaaagcgcc aacggccgat cttgaagatg atcgcccttc
      781 tctgccggat gaagtggcac tcggcgtgac ctatgacaat atcgacgact atctggaagg
      841 gaaaaacgta cctgaacagg tcgccagaac aatagagaac tggtatctga aaaccgaaca
      901 taaacgccgt ccgccaatta ccgttttcga tgatttctgg aaaaagtaat tgttttaccg
      961 agcactagtc atgaccaaaa tcccttaacg tgagtttctg ttcaccgtta catatcaaag
     1021 ggaaaacgaa tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa agcaggcaag
     1081 acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc accttgacag ctagctcagt
     1141 cctaggtata atgctagcct aacttgccat cttcaaacag gagggctgga agaagcagac
     1201 cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag tttgcaaaac
     1261 aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact caagcgtttg
     1321 cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat attacacgcc
     1381 atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atatcaagtt cctgaattcg
     1441 attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg gacagctggc
     1501 cattacaaaa cgctgacggc actgtcgcaa actatcacgg ctaccacatc gtctttgcat
     1561 tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat caaaaagtcg
     1621 gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat
     1681 tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt tcagccacat
     1741 ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa cattacggca
     1801 aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct ttgaacatca
     1861 acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat caaaatgtac
     1921 agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg agagatcctc
     1981 actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact ggaactgaag
     2041 atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa agcacatcat
     2101 tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg gctgagttag
     2161 caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa aaagtgatga
     2221 aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac gtctttaaaa
     2281 tgaacggcaa atggtatctg ttcactgact cccgcggatc aaaaatgacg attgacggca
     2341 ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact ggcccataca
     2401 agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac gatgtaacct
     2461 ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg attacaagct
     2521 atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcca agcttcctgc
     2581 tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa caaggacaat
     2641 taacagttaa caaataattt tcgcatttat cgtgaaacgc ttgtccatac ccatggattc
     2701 ttcgtctgtt tctactggta ttggcacaaa cctgattcca atttgagcaa ggctatgtgc
     2761 catctcgata ctcgttctta actcaacaga agatgctttg tgcatacagc ccctcgttta
     2821 ttatttatct cctcagccag ccgctgtgct ttcagtggat ttcggataac agaaaggccg
     2881 ggaaataccc agcctcgctt tgtaacggag tagacgaaag tgattgcgcc tacccggata
     2941 ttatcgtgag gatgcgtcat cgccattgct ccccaaatac aaaaccaatt tcagccagtg
     3001 cctcgtccat tttttcgatg aactccggca cgatctcgtc aaaactcgcc atgtactttt
     3061 catcccgctc aatcacgaca taatgcaggc cttcacgctt catacgcggg tcatagttgg
     3121 caaagtacca ggcatttttt cgcgtcaccc acatgctgta ctgcacctgg gccatgtaag
     3181 ctgactttat ggcctcgaaa ccaccgagcc ggaacttcat gaaatcccgg gaggtaaacg
     3241 ggcatttcag ttcaaggccg ttgccgtcac tgcataaacc atcgggagag caggcggtac
     3301 gcatactttc gtcgcgatag atgatcgggg attcagtaac attcacgccg gaagtgaatt
     3361 caaacagggt tctggcgtcg ttctcgtact gttttcccca ggccagtgct ttagcgttaa
     3421 cttccggagc cacaccggtg caaacctcag caagcagggt gtggaagtag gacattttca
     3481 tgtcaggcca cttctttccg gagcggggtt ttgctatcac gttgtgaact tctgaagcgg
     3541 tgatgacgcc gagccgtaat ttgtgccacg catcatcccc ctgttcgaca gctctcacat
     3601 cgatcccggt acgctgcagg ataatgtccg gtgtcatgct gccaccttct gctctgcggc
     3661 tttctgtttc aggaatccaa gagcttttac tgcttcggcc tgtgtcagtt ctgacgatgc
     3721 acgaatgtcg cggcgaaata tctgggaaca gagcggcaat aagtcgtcat cccatgtttt
     3781 atccagggcg atcagcagag tgttaatctc ctgcatggtt tcatcgttaa ccggagtgat
     3841 gtcgcgttcc ggctgacgtt ctgcagtgta tgcagtattt tcgacaatgc gctcggcttc
     3901 atccttgtca tagataccag caaatccgaa ggccagacgg gcacactgaa tcatggcttt
     3961 atgacgtaac atccgtttgg gatgcgactg ccacggcccc gtgatttctc tgccttcgcg
     4021 agttttgaat ggttcgcggc ggcattcatc catccattcg gtaacgcaga tcggatgatt
     4081 acggtccttg cggtaaatcc ggcatgtaca ggattcattg tcctgctcaa agtccatgcc
     4141 atcaaactgc tggttttcat tgatgatgcg ggaccagcca tcaacgccca ccaccggaac
     4201 gatgccattc tgcttatcag gaaaggcgta aatttctttc gtccacggat taaggccgta
     4261 ctggttggca acgatcagta atgcgatgaa ctgcgcatcg ctggcatcac ctttaaatgc
     4321 cgtctggcga agagtggtga tcagttcctg tgggtcgaca gaatccatgc cgacacgttc
     4381 agccagcttc ccagccagcg ttgcgagtgc agtactcatt cgttttatac ctctgaatca
     4441 atatcaacct ggtggtgagc aatggtttca accatgtacc ggatgtgttc tgccatgcgc
     4501 tcctgaaact caacatcgtc atcaaacgca cgggtaatgg attttttgct ggccccgtgg
     4561 cgttgcaaat gatcgatgca tagcgattca aacaggtgct ggggcaggcc tttttccatg
     4621 tcgtctgcca gttctgcctc tttctcttca cgggcgagct gctggtagtg acgcgcccag
     4681 ctctgagcct caagacgatc ctgaatgtaa taagcgttca tggctgaact cctgaaatag
     4741 ctgtgaaaat atcgcccgcg aaatgccggg ctgattagga aaacaggaaa gggggttagt
     4801 gaatgctttt gcttgatctc agtttcagta ttaatatcca ttttttataa cctccttaga
     4861 gctcgaattc ccaaaaaaac gggtatggag aaacagtaga gagttgcgat aaaaagcgtc
     4921 aggtaggatc cgctaatctt atggataaaa atgctatggc atagcaaagt gtgacgccgt
     4981 gcaaataatc aatgtggact tttctgccgt gattatagac acttttgtta cgcgtttttg
     5041 tcatggcttt ggtcccgctt tgttacagaa tgcttttaat aagcggggtt accggtttgg
     5101 ttagcgagaa gagccagtaa aagacgcagt gacggcaatg tctgatgcaa tatggacaat
     5161 tggtttcttc tctgaatggc gggagtatga aaagtatggc tgaagcgcaa aatgatcccc
     5221 tgctgccggg atactcgttt aatgcccatc tggtggcggg tttaacgccg attgaggcca
     5281 acggttatct cgattttttt atcgaccgac cgctgggaat gaaaggttat attctcaatc
     5341 tcaccattcg cggtcagggg gtggtgaaaa atcagggacg agaatttgtt tgccgaccgg
     5401 gtgatatttt gctgttcccg ccaggagaga ttcatcacta cggtcgtcat ccggaggctc
     5461 gcgaatggta tcaccagtgg gtttactttc gtccgcgcgc ctactggcat gaatggctta
     5521 actggccgtc aatatttgcc aatacggggt tctttcgccc ggatgaagcg caccagccgc
     5581 atttcagcga cctgtttggg caaatcatta acgccgggca aggggaaggg cgctattcgg
     5641 agctgctggc gataaatctg cttgagcaat tgttactgcg gcgcatggaa gcgattaacg
     5701 agtcgctcca tccaccgatg gataatcggg tacgcgaggc ttgtcagtac atcagcgatc
     5761 acctggcaga cagcaatttt gatatcgcca gcgtcgcaca gcatgtttgc ttgtcgccgt
     5821 cgcgtctgtc acatcttttc cgccagcagt tagggattag cgtcttaagc tggcgcgagg
     5881 accaacgtat cagccaggcg aagctgcttt tgagcaccac ccggatgcct atcgccaccg
     5941 tcggtcgcaa tgttggtttt gacgatcaac tctacttctc gcgggtattt aaaaaatgca
     6001 ccggggccag cccgagcgag ttccgtgccg gttgtgaaga aaaagtgaat gatgtagccg
     6061 tcaagttgtc ataataaatc gatgcaggtg gcacttttcg gggaaatgtg ccggatcccc
     6121 ctcaagtcaa aagcctccgg tcggaggttt ttgactttct gctatggagg tcaggtatga
     6181 tttaaatggt cagtattgag cgatatctag agaattcgtc aacgaattca agcttaaggt
     6241 tgaaaaataa aaacggcgct aaaaagcgcc gttttttttg acggtggtat taagacccac
     6301 tttcacattt aagttgtttt tctaatccgc atatgatcaa ttcaaggccg aataagaagg
     6361 ctggctctgc accttggtga tcaaataatt cgatagcttg tcgtaataat ggcggcatac
     6421 tatcagtagt aggtgtttcc ctttcttctt tagcgacttg atgctcttga tcttccaata
     6481 cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata taatgcattc tctagtgaaa
     6541 aaccttgttg gcataaaaag gctaattgat tttcgagagt ttcatactgt ttttctgtag
     6601 gccgtgtacc taaatgtact tttgctccat cgcgatgact tagtaaagca catctaaaac
     6661 ttttagcgtt attacgtaaa aaatcttgcc agctttcccc ttctaaaggg caaaagtgag
     6721 tatggtgcct atctaacatc tcaatggcta aggcgtcgag caaagcccgc ttatttttta
     6781 catgccaata caatgtaggc tgctctacac ctagcttctg ggcgagttta cgggttgtta
     6841 aaccttcgat tccgacctca ttaagcagct ctaatgcgct gttaatcact ttacttttat
     6901 ctaatctaga catattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6961 cgcgaaaggt tttgcgccat tcgatggtgt cctcgagttg gtaacgaatc agacaattga
     7021 cggcttgacg gagtagcata gctcgagtcc ctatcagtga tagagattga catccctatc
     7081 agtgatagag atactgagca cgagcgcacg agggagcttc cgttttagag ctagaaatag
     7141 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     7201 ttttgaagct tgggcccgaa caaaaactca tctcagaaga ggatctgaat agcgccgtcg
     7261 accatcatca tcatcatcat tgagtttaaa cggtctccag cttggctgtt ttggcggatg
     7321 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
     7381 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     7441 gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
     7501 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     7561 tgtcggtgaa ctggatcctt actcgagtct agactgcagt tgaattcgcg gccgcttcta
     7621 gagtttacgg ctagctcagt cctaggtact atgctagcta tcagcaggac gcactgacca
     7681 ggaggtacaa tcaatggata agaaatactc aataggctta gatatcggca caaatagcgt
     7741 cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg
     7801 aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg
     7861 agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa
     7921 gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag
     7981 tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca
     8041 tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta
     8101 tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt
     8161 ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc
     8221 tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt
     8281 tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt
     8341 gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg
     8401 cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt
     8461 tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga
     8521 taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt
     8581 atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc
     8641 cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa
     8701 agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa
     8761 aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat
     8821 caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga
     8881 agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt
     8941 gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa
     9001 tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc
     9061 gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg
     9121 gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac
     9181 aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt tgctttatga
     9241 gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa
     9301 accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct tcaaaacaaa
     9361 tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag aatgttttga
     9421 tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta cctaccatga
     9481 tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg aagatatctt
     9541 agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg aggaaagact
     9601 taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta
     9661 tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata agcaatctgg
     9721 caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct
     9781 gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag tgtctggaca
     9841 aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta ttaaaaaagg
     9901 tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc ggcataagcc
     9961 agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg gccagaaaaa
    10021 ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa gtcagattct
    10081 taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc tctattatct
    10141 ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt taagtgatta
    10201 tgatgtcgat cacattgttc cacaaagttt ccttaaagac gattcaatag acaataaggt
    10261 cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg aagaagtagt
    10321 caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca ctcaacgtaa
    10381 gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata aagctggttt
    10441 tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac aaattttgga
    10501 tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg ttaaagtgat
    10561 taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct ataaagtacg
    10621 tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg ttggaactgc
    10681 tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt ataaagttta
    10741 tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata
    10801 tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg caaatggaga
    10861 gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg tctgggataa
    10921 agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca atattgtcaa
    10981 gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa aaagaaattc
    11041 ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg gttttgatag
    11101 tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa
    11161 gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt cctttgaaaa
    11221 aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag acttaatcat
    11281 taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga tgctggctag
    11341 tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg tgaatttttt
    11401 atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca
    11461 attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt
    11521 ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa
    11581 acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac
    11641 gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata
    11701 tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta
    11761 tgaaacacgc attgatttga gtcagctagg aggtgacggc agcggtgatt ataaagatga
    11821 tgatgataaa tgagatatca ttcaggacga gcctcagact ccagcgtaac tggactggcg
    11881 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
    11941 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
    12001 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
    12061 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
    12121 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
    12181 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
    12241 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
    12301 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
    12361 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
    12421 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
    12481 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
    12541 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
    12601 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
    12661 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
    12721 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
    12781 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
    12841 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
    12901 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
    12961 agttttcgtt ccactgagcg tcagaccccg ttgatgatac cgctgcctta ctgggtgcat
    13021 tagccagtct gaatgacctg tcacgggata atccgaagtg gtcagactgg aaaatcagag
    13081 ggcaggaact gctgaacagc aaaaagtcag atagcaccac atagcagacc cgccataaaa
    13141 cgccctgaga agcccgtgac gggcttttct tgtattatgg gtagtttcct tgcatgaatc
    13201 cataaaaggc gcctgtagtg ccatttaccc ccattcactg ccagagccgt gagcgcagcg
    13261 aactgaatgt cacgaaaaag acagcgactc aggtgcctga tggtcggaga caaaaggaat
    13321 attcagcgat ttgcccgatt gcggccgcaa ccgagcttgc gagggtgcta cttaagcctt
    13381 tagggtttta aggtctgttt tgtagaggag caaacagcgt ttgcgacatc cttttgtaat
    13441 actgcggaac tgactaaagt agtgagttat acacagggct gggatctatt ctttttatct
    13501 ttttttattc tttctttatt ctataaatta taaccacttg aatataaaca aaaaaaacac
    13561 acaaaggtct agcggaattt acagagggtc tagcagaatt tacaagtttt ccagcaaagg
    13621 tctagcagaa tttacagata cccacaactc aaaggaaaag gactagtaat tatcattgac
    13681 tagcccatct caattggtat agtgattaaa atcacctaga ccaattgaga tgtatgtctg
    13741 aattagttgt tttcaaagca aatgaactag cgattagtcg ctatgactta acggagcatg
    13801 aaaccaagct aattttatgc tgtgtggcac tactcaaccc cacgattgaa aaccctacaa
    13861 ggaaagaacg gacggtatcg ttcacttata accaatacgt tcagatgatg aacatcagta
    13921 gggaaaatgc ttatggtgta ttagctaaag caaccagaga gctgatgacg agaactgtgg
    13981 aaatcaggaa tcctttggtt aaaggctttg agattttcca gtggacaaac tatgccaagt
    14041 tctcaagcga aaaattagaa ttagttttta gtgaagagat attgccttat cttttccagt
    14101 taaaaaaatt cataaaatat aatctggaac atgttaagtc ttttgaaaac aaatactcta
    14161 tgaggattta tgagtggtta ttaaaagaac taacacaaaa gaaaactcac aaggcaaata
    14221 tagagattag ccttgatgaa tttaagttca tgttaatgct tgaaaataac taccatgagt
    14281 ttaaaaggct taaccaatgg gttttgaaac caataagtaa agatttaaac acttacagca
    14341 atatgaaatt ggtggttgat aagcgaggcc gcccgactga tacgttgatt ttccaagttg
    14401 aactagatag acaaatggat ctcgtaaccg aacttgagaa caaccagata aaaatgaatg
    14461 gtgacaaaat accaacaacc attacatcag attcctacct acataacgga ctaagaaaaa
    14521 cactacacga tgctttaact gcaaaaattc agctcaccag ttttgaggca aaatttttga
    14581 gtgacatgca aagtaagtat gatctcaatg gttcgttctc atggctcacg caaaaacaac
    14641 gaaccacact agagaacata ctggctaaat acggaaggat ctgag
//
