LOCUS       sequence-486997-       10868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For affinity purification of Pvc10 fused to Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10868)
  AUTHORS   Kreitz J, Yang V, Friedrich MJ, Pham J, Macrae RK, Zhang F
  TITLE     Targeted delivery of diverse biomolecules with engineered bacterial 
            nanosyringes.
  JOURNAL   Nat Biotechnol. 2025 Aug 12. doi: 10.1038/s41587-025-02774-x.
  PUBMED    40796978
REFERENCE   2  (bases 1 to 10868)
  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: "Nat 
            Biotechnol. 2025 Aug 12. doi: 10.1038/s41587-025-02774-x."
COMMENT     Sequence Label: TwinStrepSUMO-pvc10-Cas9
FEATURES             Location/Qualifiers
     source          1..10868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2079..2098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2239..2261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2255..2446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3758..4840)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4802..4821
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4841..4918)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5124..5143
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5227..5246
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5227..5245
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5246..5270
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             5285..5307
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5326..5343
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5353..5370
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5383..5406,5407..5442,5443..5466)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             5467..5760
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             6277..6342
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6352..6372
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6397..10500
                     /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             6397..10500
                     /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"
     primer_bind     complement(10595..10613)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      10609..10656
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(10831..10836,10837..10853,10854..10859))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2101 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2281 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2341 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2401 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2461 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2521 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2581 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2641 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     2701 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     2761 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     2821 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     2881 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     2941 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     3001 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     3061 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     3121 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     3181 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     3241 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     3301 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     3361 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     3421 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3481 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3541 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3601 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3661 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3721 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3781 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3841 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     3901 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     3961 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     4021 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     4081 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     4141 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     4201 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4261 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4321 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4381 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4441 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4501 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4561 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4621 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4681 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4741 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     4801 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     4861 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     4921 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     4981 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     5041 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     5101 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     5161 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     5221 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     5281 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
     5341 cacagcagcg gcctggtgcc gcgcggcagc catatggcta gctggagcca tccgcagttt
     5401 gaaaaaggtg gtggtagcgg tggtggttca ggtggtagtg catggtcaca ccctcagttt
     5461 gagaaaatgt cggactcaga agtcaatcaa gaagctaagc cagaggtcaa gccagaagtc
     5521 aagcctgaga ctcacatcaa tttaaaggtg tccgatggat cttcagagat cttcttcaag
     5581 atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag acagggtaag
     5641 gaaatggact ccttaagatt cttgtacgac ggtattagaa tccaagctga tcagacccct
     5701 gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca gattggtgga
     5761 tccatgagtg aagcgattgt ggtggatggt gacgtgttac agtttgatcc caactttggc
     5821 aatcggcagg tgacggttcc cagcccagga aaaattagcg gcacaggaca tgcgcaggta
     5881 agtggaaaaa aagtgtgtat tctgggggat gagaaacagg tcagggtttc tgcaacctat
     5941 attacaacaa cacatactac gccgggaaca ggaaccatta ctatcagtgc tctggatgct
     6001 ggccagcagg cccttcagtg taccagtggg gcggctttaa ttatcaaggg gcagcaattt
     6061 acggcgatgt ttacgcctga attgccagcc atgaataata cagtgactcc gccacaaccg
     6121 gatgttacga caccttcatc aggaaaagga cgttttatca ctcaacaaaa ttttgctacc
     6181 gtaaatggtg gttcaggtgg tggtggtagc ggtggtggcg gttctggcgg tggcggtagt
     6241 ggcggaggtg gaagcggagg cggtggcagt ggtggtgatt ataaagatca tgatggcgac
     6301 tacaaagacc acgacattga ttacaaagat gacgacgaca aaggtggcgc accgaaaaag
     6361 aaacgcaaag ttggtattca tggtgttccg gcagcaatgg ataagaaata ctcaataggc
     6421 ttagatatcg gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaggttccg
     6481 tctaaaaagt tcaaggttct gggaaataca gaccgccaca gtatcaaaaa aaatcttata
     6541 ggggctcttt tatttgacag tggagagaca gcggaagcga ctcgtctcaa acggacagct
     6601 cgtagaaggt atacacgtcg gaagaatcgt atttgttatc tacaggagat tttttcaaat
     6661 gagatggcga aagtagatga tagtttcttt catcgacttg aagagtcttt tttggtggaa
     6721 gaagacaaga agcatgaacg tcatcctatt tttggaaata tagtagatga agttgcttat
     6781 catgagaaat atccaactat ctatcatctg cgaaaaaaat tggtagattc tactgataaa
     6841 gcggatttgc gcttaatcta tttggcctta gcgcatatga ttaagtttcg tggtcatttt
     6901 ttgattgagg gagatttaaa tcctgataat agtgatgtgg acaaactatt tatccagttg
     6961 gtacaaacct acaatcaatt atttgaagaa aaccctatta acgcaagtgg agtagatgct
     7021 aaagcgattc tttctgcacg attgagtaaa tcaagacgat tagaaaatct cattgctcag
     7081 ctccccggtg agaagaaaaa tggcttattt gggaatctca ttgctttgtc attgggtttg
     7141 acccctaatt ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca gctttcaaaa
     7201 gatacttacg atgatgattt agataattta ttggcgcaaa ttggagatca atatgctgat
     7261 ttgtttttgg cagctaagaa tttatcagat gctattttac tttcagatat cctaagagta
     7321 aatactgaaa taactaaggc tcccctatca gcttcaatga ttaaacgcta cgatgaacat
     7381 catcaagact tgactctttt aaaagcttta gttcgacaac aacttccaga aaagtataaa
     7441 gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg gggagctagc
     7501 caagaagaat tttataaatt tatcaaacca attttagaaa aaatggatgg tactgaggaa
     7561 ttattggtga aactaaatcg tgaagatttg ctgcgcaagc aacggacctt tgacaacggc
     7621 tctattcccc atcaaattca cttgggtgag ctgcatgcta ttttgagaag acaagaagac
     7681 ttttatccat ttttaaaaga caatcgtgag aagattgaaa aaatcttgac ttttcgaatt
     7741 ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag
     7801 tctgaagaaa caattacccc atggaatttt gaagaagttg tcgataaagg tgcttcagct
     7861 caatcattta ttgaacgcat gacaaacttt gataaaaatc ttccaaatga aaaagtacta
     7921 ccaaaacata gtttgcttta tgagtatttt acggtttata acgaattgac aaaggtcaaa
     7981 tatgttactg aaggaatgcg aaaaccagca tttctttcag gtgaacagaa gaaagccatt
     8041 gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa agaagattat
     8101 ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag gagttgaaga tagatttaat
     8161 gcttcattag gtacctacca tgatttgcta aaaattatta aagataaaga ttttttggat
     8221 aatgaagaaa atgaagatat cttagaggat attgttttaa cattgacctt atttgaagat
     8281 agggagatga ttgaggaaag acttaaaaca tatgctcacc tctttgatga taaggtgatg
     8341 aaacagctta aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa attgattaat
     8401 ggtattaggg ataagcaatc tggcaaaaca atattagatt ttttgaaatc agatggtttt
     8461 gccaatcgca attttatgca gctgatccat gatgatagtt tgacatttaa agaagacatt
     8521 caaaaagcac aagtgtctgg acaaggcgat agtttacatg aacatattgc aaatttagct
     8581 ggtagccctg ctattaaaaa aggtatttta cagactgtaa aagttgttga tgaattggtc
     8641 aaagtaatgg ggcggcataa gccagaaaat atcgttattg aaatggcacg tgaaaatcag
     8701 acaactcaaa agggccagaa aaattcgcga gagcgtatga aacgaatcga agaaggtatc
     8761 aaagaattag gaagtcagat tcttaaagag catcctgttg aaaatactca attgcaaaat
     8821 gaaaagctct atctctatta tctccaaaat ggaagagaca tgtatgtgga ccaagaatta
     8881 gatattaatc gtttaagtga ttatgatgtc gatcacattg ttccacaaag tttccttaaa
     8941 gacgattcaa tagacaataa ggtcttaacg cgttctgata aaaatcgtgg taaatcggat
     9001 aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact attggagaca acttctaaac
     9061 gccaagttaa tcactcaacg taagtttgat aatttaacga aagctgaacg tggaggtttg
     9121 agtgaacttg ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact
     9181 aagcatgtgg cacaaatttt ggatagtcgc atgaatacta aatacgatga aaatgataaa
     9241 cttattcgag aggttaaagt gattacctta aaatctaaat tagtttctga cttccgaaaa
     9301 gatttccaat tctataaagt acgtgagatt aacaattacc atcatgccca tgatgcgtat
     9361 ctaaatgccg tcgttggaac tgctttgatt aagaaatatc caaaacttga atcggagttt
     9421 gtctatggtg attataaagt ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa
     9481 ataggcaaag caaccgcaaa atatttcttt tactctaata tcatgaactt cttcaaaaca
     9541 gaaattacac ttgcaaatgg agagattcgc aaacgccctc taatcgaaac taatggggaa
     9601 actggagaaa ttgtctggga taaagggcga gattttgcca cagtgcgcaa agtattgtcc
     9661 atgccccaag tcaatattgt caagaaaaca gaagtacaga caggcggatt ctccaaggag
     9721 tcaattttac caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga ctgggatcca
     9781 aaaaaatatg gtggttttga tagtccaacg gtagcttatt cagtcctagt ggttgctaag
     9841 gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag agttactagg gatcacaatt
     9901 atggaaagaa gttcctttga aaaaaatccg attgactttt tagaagctaa aggatataag
     9961 gaagttaaaa aagacttaat cattaaacta cctaaatata gtctttttga gttagaaaac
    10021 ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa aaggaaatga gctggctctg
    10081 ccaagcaaat atgtgaattt tttatattta gctagtcatt atgaaaagtt gaagggtagt
    10141 ccagaagata acgaacaaaa acaattgttt gtggagcagc ataagcatta tttagatgag
    10201 attattgagc aaatcagtga attttctaag cgtgttattt tagcagatgc caatttagat
    10261 aaagttctta gtgcatataa caaacataga gacaaaccaa tacgtgaaca agcagaaaat
    10321 attattcatt tatttacgtt gacgaatctt ggagctcccg ctgcttttaa atattttgat
    10381 acaacaattg atcgtaaacg atatacgtct acaaaagaag ttttagatgc cactcttatc
    10441 catcaatcca tcactggtct ttatgaaaca cgcattgatt tgagtcagct aggaggtgac
    10501 aaacgtccgg cagcaaccaa aaaggcaggt caggccaaaa agaaatgagc ggccacactc
    10561 gaggcccgaa aggaagctaa gttggctgct gccaccgctg agcaataact agcataaccc
    10621 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
    10681 tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg
    10741 tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc
    10801 gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa
    10861 catgagaa
//
