LOCUS       sequence-356882-        7490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses spCas9 and gRNA that targets ColE1 plasmids for curing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 3730 to 3764)
  AUTHORS   Lee KZ, Mechikoff MA, Parasa MK, Rankin TJ, Pandolfi P, Fitzgerald 
            KS, Hillman ET, Solomon KV
  TITLE     Repurposing the Homing Endonuclease I-SceI for Positive Selection 
            and Development of Gene-Editing Technologies.
  JOURNAL   ACS Synth Biol. 2022 Jan 21;11(1):53-60. doi: 
            10.1021/acssynbio.1c00340. Epub 2022 Jan 10.
  PUBMED    35007422
REFERENCE   2  (bases 1 to 7490)
  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"; doi: 
            "10.1021/acssynbio.1c00340"; journalName: "ACS Synth Biol"; date: 
            "2022-01-21- 21"; volume: "11"; issue: "1"; pages: "53-60"
COMMENT     Sequence Label: pFREE2-SpCas9-sgRNA
FEATURES             Location/Qualifiers
     source          1..7490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(862..880)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      876..923
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1057..1680
                     /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(1130..1149)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(1842..2657)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1842..2657)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2565..2584
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2658..2749)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2767..3402)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(2912..2931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(3651..3729)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(3730..3764)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     repeat_region   3907..3942
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3973..4008
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     protein_bind    4082..4100
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4107..4125
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4211..7490,1..827)
                     /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             join(4211..7490,1..824)
                     /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"
ORIGIN
        1 ttgtcaagaa aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa
       61 gaaattcgga caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt
      121 ttgatagtcc aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat
      181 cgaagaagtt aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct
      241 ttgaaaaaaa tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact
      301 taatcattaa actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc
      361 tggctagtgc cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga
      421 attttttata tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac
      481 aaaaacaatt gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca
      541 gtgaattttc taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat
      601 ataacaaaca tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta
      661 cgttgacgaa tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta
      721 aacgatatac gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg
      781 gtctttatga aacacgcatt gatttgagtc agctaggagg tgactgagat cccatgttac
      841 cggtatccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
      901 aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac
      961 tgcttccggt agtcaataaa ccggtattga cgccaacggg tcacacgggt acagtgctag
     1021 cagaaacata tccatgaaat cccgccccga attcatatgt ctagattaga taaaagtaaa
     1081 gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt
     1141 aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag
     1201 cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc
     1261 cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt
     1321 gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa
     1381 cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag
     1441 aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat
     1501 caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca
     1561 ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc
     1621 ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtcttaa
     1681 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc
     1741 gggtctccgc aagtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1801 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1861 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1921 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1981 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2041 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2101 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2161 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2221 acgcggtcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2281 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2341 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2401 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2461 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2521 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2581 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2641 cgttgaatat ggctcatact cttccttttt caatattatt gaagcattta tcagggttat
     2701 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggcatgcta
     2761 gcgcagaaac gtcctagaag atgccaggag gatacttagc agagagacaa taaggccgga
     2821 gcgaagccgt ttttccatag gctccgcccc cctgacgaac atcacgaaat ctgacgctca
     2881 aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctgatggc
     2941 tccctcttgc gctctcctgt tcccgtcctg cggcgtccgt gttgtggtgg aggctttacc
     3001 caaatcacca cgtcccgttc cgtgtagaca gttcgctcca agctgggctg tgtgcaagaa
     3061 ccccccgttc agcccgactg ctgcgcctta tccggtaact atcatcttga gtccaacccg
     3121 gaaagacacg acaaaacgcc actggcagca gccattggta actgagaatt agtggattta
     3181 gatatcgaga gtcttgaagt ggtggcctaa cagaggctac actgaaagga cagtatttgg
     3241 tatctgcgct ccactaaagc cagttaccag gttaagcagt tccccaactg acttaacctt
     3301 cgatcaaacc gcctccccag gcggtttttt cgtttacaga gcaggagatt acgacgatcg
     3361 taaaaggatc tcaagaagat cctttacgga ttcccgacac catcactcta gatttcagtg
     3421 caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc
     3481 tcatgttagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     3541 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgca
     3601 caccgactag cgaaaaaacc ccgccgaagc ggggtttttt gcgaaaaaaa gcaccgactc
     3661 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tgctgttttg
     3721 aatggttccg ctagcactgt acctaggact gagctagccg tcaaaagcag cgtatatacc
     3781 actgcagtgg ccacaattca gcaaattgtg aacatcatca cgttcatctt tccctggttg
     3841 ccaatggccc attttcctgt cagtaacgag aaggtcgcga attcaggcgc tttttagact
     3901 ggtcgtgttt tagagctatg ctgttttgaa tggtcccaaa actgcggcga gcggtatcag
     3961 ctcactcaaa gggttttaga gctatgctgt tttgaatggt cccaaaacaa aaaaaaaccc
     4021 cgcccctgac agggcggggt ttttttttta attaaagtct ggtcctcgag tctggttata
     4081 atccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg agcacagaag
     4141 gagatataca tggataagaa atactgagca ctatcagtga tagagatact gagcacagaa
     4201 ggagatatac atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg
     4261 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
     4321 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
     4381 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
     4441 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
     4501 ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc
     4561 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
     4621 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
     4681 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
     4741 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
     4801 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
     4861 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
     4921 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
     4981 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
     5041 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
     5101 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
     5161 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
     5221 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
     5281 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
     5341 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
     5401 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
     5461 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg
     5521 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg
     5581 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
     5641 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
     5701 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
     5761 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
     5821 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
     5881 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
     5941 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
     6001 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
     6061 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
     6121 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
     6181 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
     6241 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
     6301 ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg
     6361 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
     6421 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
     6481 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
     6541 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
     6601 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
     6661 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
     6721 tgtcgatcac attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
     6781 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
     6841 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
     6901 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
     6961 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
     7021 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
     7081 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
     7141 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
     7201 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
     7261 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
     7321 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
     7381 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
     7441 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata
//
