LOCUS       sequence-333574-       11215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 and beta expression plasmid for performing gene knockouts via 
            oligo recombineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11215)
  AUTHORS   Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez 
            D, Thomas MG, Pfleger BF
  TITLE     Stepwise genetic engineering of Pseudomonas putida enables robust 
            heterologous production of prodigiosin and glidobactin A.
  JOURNAL   Metab Eng. 2021 Jun 24;67:112-124. doi: 10.1016/j.ymben.2021.06.004.
  PUBMED    34175462
REFERENCE   2  (bases 1 to 11215)
  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: "Metab 
            Eng."; date: "2021-06-24"; volume: "67:112-124. doi"; pages: " 
            10.1016/j.ymben.2021.06.004"
COMMENT     Sequence Label: pCas9-beta
FEATURES             Location/Qualifiers
     source          1..11215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      94..140
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    190..211
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        212..246
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     CDS             282..4385
                     /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             282..4385
                     /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             4395..4418
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        join(4633..4638,4639..4655,4656..4661)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4850..5383
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEVVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     CDS             6624..7772
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(7859..8644)
                     /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"
     RBS             8653..8661
                     /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(8688..8972)
                     /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(8746..8765)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             8999..9877
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     terminator      complement(9928..9959)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(10093..10716)
                     /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     10624..10643
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(10717..10794)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    10851..10869
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10876..10894
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        10925..11000
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             11021..11050
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             11066..11083
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(11139..11156)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
       61 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
      121 gactgggcct ttcgttttat ctgttgtttg tcggtgaact ggatccttac tcgagtctag
      181 actgcagttg aattcgcggc cgcttctaga gtttacggct agctcagtcc taggtactat
      241 gctagctatc agcaggacgc actgaccagg aggtacaatc aatggataag aaatactcaa
      301 taggcttaga tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg
      361 ttccgtctaa aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc
      421 ttataggggc tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga
      481 cagctcgtag aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt
      541 caaatgagat ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg
      601 tggaagaaga caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg
      661 cttatcatga gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg
      721 ataaagcgga tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc
      781 attttttgat tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc
      841 agttggtaca aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag
      901 atgctaaagc gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg
      961 ctcagctccc cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg
     1021 gtttgacccc taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt
     1081 caaaagatac ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg
     1141 ctgatttgtt tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa
     1201 gagtaaatac tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg
     1261 aacatcatca agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt
     1321 ataaagaaat cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag
     1381 ctagccaaga agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg
     1441 aggaattatt ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca
     1501 acggctctat tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag
     1561 aagactttta tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc
     1621 gaattcctta ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc
     1681 ggaagtctga agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt
     1741 cagctcaatc atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag
     1801 tactaccaaa acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg
     1861 tcaaatatgt tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag
     1921 ccattgttga tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag
     1981 attatttcaa aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat
     2041 ttaatgcttc attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt
     2101 tggataatga agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg
     2161 aagataggga gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg
     2221 tgatgaaaca gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga
     2281 ttaatggtat tagggataag caatctggca aaacaatatt agattttttg aaatcagatg
     2341 gttttgccaa tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag
     2401 acattcaaaa agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt
     2461 tagctggtag ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat
     2521 tggtcaaagt aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa
     2581 atcagacaac tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag
     2641 gtatcaaaga attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc
     2701 aaaatgaaaa gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag
     2761 aattagatat taatcgttta agtgattatg atgtcgatca cattgttcca caaagtttcc
     2821 ttaaagacga ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat
     2881 cggataacgt tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc
     2941 taaacgccaa gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag
     3001 gtttgagtga acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa
     3061 tcactaagca tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg
     3121 ataaacttat tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc
     3181 gaaaagattt ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg
     3241 cgtatctaaa tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg
     3301 agtttgtcta tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc
     3361 aagaaatagg caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca
     3421 aaacagaaat tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg
     3481 gggaaactgg agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat
     3541 tgtccatgcc ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca
     3601 aggagtcaat tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg
     3661 atccaaaaaa atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg
     3721 ctaaggtgga aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca
     3781 caattatgga aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat
     3841 ataaggaagt taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag
     3901 aaaacggtcg taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg
     3961 ctctgccaag caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg
     4021 gtagtccaga agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag
     4081 atgagattat tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt
     4141 tagataaagt tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag
     4201 aaaatattat tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt
     4261 ttgatacaac aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc
     4321 ttatccatca atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag
     4381 gtgacggcag cggtgattat aaagatgatg atgataaatg agatatcatt caggacgagc
     4441 ctcagactcc agcgtaactg gactgcgcat gcataaaaac tgttgtaatt cattaagcat
     4501 tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag
     4561 caccttgtcg ccttgcgtat aatatttgcc catggacgca caccgtggaa acggatgaag
     4621 gcacgaaccc agttgacata agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg
     4681 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
     4741 gttttcatgg cttgttatga ctgttttttt gtacagtcta tgcctcgggc atccaagcag
     4801 caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag
     4861 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg gctcaagtat
     4921 gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc gggctgctct
     4981 tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc agccggactc
     5041 cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg ccttcgacca
     5101 agaagtggtt gttggcgctc tcgcggctta cgttctgccc aagtttgagc agccgcgtag
     5161 tgagatctat atctatgatc tcgcagtctc cggagagcac cggaggcagg gcattgccac
     5221 cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg tgatctacgt
     5281 gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg gcatacggga
     5341 agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg ttcaagccga
     5401 gatcggcttc ccggccgcgg agttgttcgg taaattgtca caacgccgca ggtggcactt
     5461 ttcggggaaa tgtgcgcgcc cgcgttcctg ctggcgctgg gcctgtttct ggcgctggac
     5521 ttcccgctgt tccgtcagca gcttttcgcc cacggccttg atgatcgcgg cggccttggc
     5581 ctgcatatcc cgattcaacg gccccagggc gtccagaacg ggcttcaggc gctcccgaag
     5641 gtggatcccc ccctcgcatt ggtaactgtc agaccaaggt gagaatccaa gcactagtgt
     5701 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgggggatca ggaccgctgc
     5761 cggagcgcaa cccactcact acagcagagc catgtagggc cgccggcgtt gtggatacct
     5821 cgcggaaaac ttggccctca ctgacagatg aggggcggac gttgacactt gaggggccga
     5881 ctcacccggc gcggcgttga cagatgaggg gcaggctcga tttcggccgg cgacgtggag
     5941 ctggccagcc tcgcaaatcg gcgaaaacgc ctgattttac gcgagtttcc cacagatgat
     6001 gtggacaagc ctggggataa gtgccctgcg gtattgacac ttgaggggcg cgactactga
     6061 cagatgaggg gcgcgatcct tgacacttga ggggcagagt gctgacagat gaggggcgca
     6121 cctattgaca tttgaggggc tgtccacagg cagaaaatcc agcatttgca agggtttccg
     6181 cccgtttttc ggccaccgct aacctgtctt ttaacctgct tttaaaccaa tatttataaa
     6241 ccttgttttt aaccagggct gcgcccctcg gccgcgaacg gcctcacccc aaaaatggca
     6301 gccacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
     6361 gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
     6421 acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     6481 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     6541 ccaaggatct gatggcgcag gggatcaaga tcgacggatc gatccgggga attaattccg
     6601 gggcaatccc gcaaggaggg tgaatgaatc ggacgtttga ccggaaggca tacaggcaag
     6661 aactgatcga cgcggggttt tccgccgagg atgccgaaac catcgcaagc cgcaccgtca
     6721 tgcgtgcgcc ccgcgaaacc ttccagtccg tcggctcgat agtccagcaa gctacggcca
     6781 agatcgagcg cgacagcgtg caactggctc cccctgccct gcccgcgcca tcggccgccg
     6841 tggagcgttc gcgtcgtctc gaacaggagg cggcaggttt ggcgaagtcg atgaccatcg
     6901 acacgcgagg aactatgacg accaagaagc gaaaaaccgc cggcgaggac ctggcaaaac
     6961 aggtcagcga agccaagcag gccgcgttgc tgaaacacac gaagcagcag atcaaggaaa
     7021 tgcagctttc cttgttcgat attgcgccgt ggccggacac gatgcgagcg atgccaaacg
     7081 acacggcccg ctctgccctg ttcaccacgc gcaacaagaa aatcccgcgc gaggcgctgc
     7141 aaaacaaggt cattttccac gtcaacaagg acgtgaagat cacctacacc ggcgtcgagc
     7201 tgcgggccga cgatgacgaa ctggtgtggc agcaggtgtt ggagtacgcg aagcgcaccc
     7261 ctatcggcga gccgatcacc ttcacgttct acgagctttg ccaggacctg ggctggtcga
     7321 tcaatggccg gtattacacg aaggccgagg aatgcctgtc gcgcctacag gcgacggcga
     7381 tgggcttcac gtccgaccgc gttgggcacc tggaatcggt gtcgctgctg caccgcttcc
     7441 gcgtcctgga ccgtggcaag aaaacgtccc gttgccaggt cctgatcgac gaggaaatcg
     7501 tcgtgctgtt tgctggcgac cactacacga aattcatatg ggagaagtac cgcaagctgt
     7561 cgccgacggc ccgacggatg ttcgactatt tcagctcgca ccgggagccg tacccgctca
     7621 agctggaaac cttccgcctc atgtgcggat cggattccac ccgcgtgaag aagtggcgcg
     7681 agcaggtcgg cgaagcctgc gaagagttgc gaggcagcgg cctggtggaa cacgcctggg
     7741 tcaatgatga cctggtgcat tgcaaacgct agggccttgt ggggtcagtt ccggctgggg
     7801 gttcagcagc cacctgcatc gccctaggta taaagggaaa actgtccata cccatggatc
     7861 atgctgccac cttctgctct gcggctttct gtttcaggaa tccaagagct tttactgctt
     7921 cggcctgtgt cagttctgac gatgcacgaa tgtcgcggcg aaatatctgg gaacagagcg
     7981 gcaataagtc gtcatcccat gttttatcca gggcgatcag cagagtgtta atctcctgca
     8041 tggtttcatc gttaaccgga gtgatgtcgc gttccggctg acgttctgca gtgtatgcag
     8101 tattttcgac aatgcgctcg gcttcatcct tgtcatagat accagcaaat ccgaaggcca
     8161 gacgggcaca ctgaatcatg gctttatgac gtaacatccg tttgggatgc gactgccacg
     8221 gccccgtgat ttctctgcct tcgcgagttt tgaatggttc gcggcggcat tcatccatcc
     8281 attcggtaac gcagatcgga tgattacggt ccttgcggta aatccggcat gtacaggatt
     8341 cattgtcctg ctcaaagtcc atgccatcaa actgctggtt ttcattgatg atgcgggacc
     8401 agccatcaac gcccaccacc ggaacgatgc cattctgctt atcaggaaag gcgtaaattt
     8461 ctttcgtcca cggattaagg ccgtactggt tggcaacgat cagtaatgcg atgaactgcg
     8521 catcgctggc atcaccttta aatgccgtct ggcgaagagt ggtgatcagt tcctgtgggt
     8581 cgacagaatc catgccgaca cgttcagcca gcttcccagc cagcgttgcg agtgcagtac
     8641 tcatttttta taacctcctt agagctcgaa ttcccaaaaa aacgggtatg gagaaacagt
     8701 agagagttgc gataaaaagc gtcaggtagg atccgctaat cttatggata aaaatgctat
     8761 ggcatagcaa agtgtgacgc cgtgcaaata atcaatgtgg acttttctgc cgtgattata
     8821 gacacttttg ttacgcgttt ttgtcatggc tttggtcccg ctttgttaca gaatgctttt
     8881 aataagcggg gttaccggtt tggttagcga gaagagccag taaaagacgc agtgacggca
     8941 atgtctgatg caatatggac aattggtttc ttctctgaat ggcgggagta tgaaaagtat
     9001 ggctgaagcg caaaatgatc ccctgctgcc gggatactcg tttaatgccc atctggtggc
     9061 gggtttaacg ccgattgagg ccaacggtta tctcgatttt tttatcgacc gaccgctggg
     9121 aatgaaaggt tatattctca atctcaccat tcgcggtcag ggggtggtga aaaatcaggg
     9181 acgagaattt gtttgccgac cgggtgatat tttgctgttc ccgccaggag agattcatca
     9241 ctacggtcgt catccggagg ctcgcgaatg gtatcaccag tgggtttact ttcgtccgcg
     9301 cgcctactgg catgaatggc ttaactggcc gtcaatattt gccaatacgg ggttctttcg
     9361 cccggatgaa gcgcaccagc cgcatttcag cgacctgttt gggcaaatca ttaacgccgg
     9421 gcaaggggaa gggcgctatt cggagctgct ggcgataaat ctgcttgagc aattgttact
     9481 gcggcgcatg gaagcgatta acgagtcgct ccatccaccg atggataatc gggtacgcga
     9541 ggcttgtcag tacatcagcg atcacctggc agacagcaat tttgatatcg ccagcgtcgc
     9601 acagcatgtt tgcttgtcgc cgtcgcgtct gtcacatctt ttccgccagc agttagggat
     9661 tagcgtctta agctggcgcg aggaccaacg tatcagccag gcgaagctgc ttttgagcac
     9721 cacccggatg cctatcgcca ccgtcggtcg caatgttggt tttgacgatc aactctactt
     9781 ctcgcgggta tttaaaaaat gcaccggggc cagcccgagc gagttccgtg ccggttgtga
     9841 agaaaaagtg aatgatgtag ccgtcaagtt gtcataataa atcgatgcag gtggcacttt
     9901 tcggggaaat gtgccggatc cccctcaagt caaaagcctc cggtcggagg tttttgactt
     9961 tctgctatgg aggtcaggta tgatttaaat ggtcagtatt gagcgatatc tagagaattc
    10021 gtcaacgaat tcaagcttaa ggttgaaaaa taaaaacggc gctaaaaagc gccgtttttt
    10081 ttgacggtgg tattaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat
    10141 caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata attcgatagc
    10201 ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt ctttagcgac
    10261 ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag cgctgagtgc
    10321 atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt gattttcgag
    10381 agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc catcgcgatg
    10441 acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt gccagctttc
    10501 cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc
    10561 gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta cacctagctt
    10621 ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca gctctaatgc
    10681 gctgttaatc actttacttt tatctaatct agacatattc accaccctga attgactctc
    10741 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtcctcgag
    10801 ttggtaacga atcagacaat tgacggcttg acggagtagc atagctcgag tccctatcag
    10861 tgatagagat tgacatccct atcagtgata gagatactga gcacgagcgc acgagggagc
    10921 ttccgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
    10981 aagtggcacc gagtcggtgc tttttttgaa gcttgggccc gaacaaaaac tcatctcaga
    11041 agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt aaacggtctc
    11101 cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
    11161 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcc
//
