LOCUS       sequence-485905-        9663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 nuclease with fused p2c targeting ligand for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9663)
  AUTHORS   Chaverra-Rodriguez D, Macias VM, Hughes GL, Pujhari S, Suzuki Y, 
            Peterson DR, Kim D, McKeand S, Rasgon JL
  TITLE     Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod 
            ovaries for heritable germline gene editing.
  JOURNAL   Nat Commun. 2018 Aug 1;9(1):3008. doi: 10.1038/s41467-018-05425-9.
  PUBMED    30068905
REFERENCE   2  (bases 1 to 9663)
  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 
            Commun."; date: "2018-08-1"; volume: "9(1):3008. doi"; pages: " 
            10.1038/s41467-018-05425-9"
COMMENT     Sequence Label: pET28(a)-p2c-Cas9
FEATURES             Location/Qualifiers
     source          1..9663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             148..180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             340..4443
                     /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             340..4443
                     /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             4459..4479
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4480..4506
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4480..4506
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4528..4545
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4598..4616)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4612..4659
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4696..5151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4783..4802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4993..5014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5243..6058)
                     /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(5243..6058)
                     /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     5966..5985
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6050..6069
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6180..6768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6669..6688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6922..6939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6954..7093
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7019..7038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7179..7201
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7195..7386)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(8195..9277)
                     /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     9239..9258
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9278..9355)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9561..9580
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatcca atctgcagca gcagcgccag cacggcaaga acggcaacca ggactaccag
      241 gatcagagca acgaacagag gaagaaccag aggaccagca gcgaggagga ctacagcgag
      301 gaggttaaga acgagctccg tcgacaagct tgcggccgca tggacaagaa gtacagcatc
      361 ggcctggaca tcggtaccaa cagcgtgggc tgggccgtga tcaccgacga gtacaaggtg
      421 cccagcaaga agttcaaggt gctgggcaac accgaccgcc acagcatcaa gaagaacctg
      481 atcggcgccc tgctgttcga cagcggcgag accgccgagg ccacccgcct gaagcgcacc
      541 gcccgccgcc gctacacccg ccgcaagaac cgcatctgct acctgcagga gatcttcagc
      601 aacgagatgg ccaaggtgga cgacagcttc ttccaccgcc tggaggagag cttcctggtg
      661 gaggaggaca agaagcacga gcgccacccc atcttcggca acatcgtgga cgaggtggcc
      721 taccacgaga agtaccccac catctaccac ctgcgcaaga agctggtgga cagcaccgac
      781 aaggccgacc tgcgcctgat ctacctggcc ctggcccaca tgatcaagtt ccgcggccac
      841 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
      901 ctggtgcaga cctacaacca gctgttcgag gagaacccca tcaacgccag cggcgtggac
      961 gccaaggcca tcctgagcgc ccgcctgagc aagagccgcc gcctggagaa cctgatcgcc
     1021 cagctgcccg gcgagaagaa gaacggcctg ttcggcaacc tgatcgccct gagcctgggc
     1081 ctgaccccca acttcaagag caacttcgac ctggccgagg acgccaagct gcagctgagc
     1141 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     1201 gacctgttcc tggccgccaa gaacctgagc gacgccatcc tgctgagcga catcctgcgc
     1261 gtgaacaccg agatcaccaa ggcccccctg agcgccagca tgatcaagcg ctacgacgag
     1321 caccaccagg acctgaccct gctgaaggcc ctggtgcgcc agcagctgcc cgagaagtac
     1381 aaggagatct tcttcgacca gagcaagaac ggctacgccg gctacatcga cggcggcgcc
     1441 agccaggagg agttctacaa gttcatcaag cccatcctgg agaagatgga cggcaccgag
     1501 gagctgctgg tgaagctgaa ccgcgaggac ctgctgcgca agcagcgcac cttcgacaac
     1561 ggcagcatcc cccaccagat ccacctgggc gagctgcacg ccatcctgcg ccgccaggag
     1621 gacttctacc ccttcctgaa ggacaaccgc gagaagatcg agaagatcct gaccttccgc
     1681 atcccctact acgtgggccc cctggcccgc ggcaacagcc gcttcgcctg gatgacccgc
     1741 aagagcgagg agaccatcac cccctggaac ttcgaggagg tggtggacaa gggcgccagc
     1801 gcccagagct tcatcgagcg catgaccaac ttcgacaaga acctgcccaa cgagaaggtg
     1861 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaggtg
     1921 aagtacgtga ccgagggcat gcgcaagccc gccttcctga gcggcgagca gaagaaggcc
     1981 atcgtggacc tgctgttcaa gaccaaccgc aaggtgaccg tgaagcagct gaaggaggac
     2041 tacttcaaga agatcgagtg cttcgacagc gtggagatca gcggcgtgga ggaccgcttc
     2101 aacgccagcc tgggcaccta ccacgacctg ctgaagatca tcaaggacaa ggacttcctg
     2161 gacaacgagg agaacgagga catcctggag gacatcgtgc tgaccctgac cctgttcgag
     2221 gaccgcgaga tgatcgagga gcgcctgaag acctacgccc acctgttcga cgacaaggtg
     2281 atgaagcagc tgaagcgccg ccgctacacc ggctggggcc gcctgagccg caagcttatc
     2341 aacggcatcc gcgacaagca gagcggcaag accatcctgg acttcctgaa gagcgacggc
     2401 ttcgccaacc gcaacttcat gcagctgatc cacgacgaca gcctgacctt caaggaggac
     2461 atccagaagg cccaggtgag cggccagggc gacagcctgc acgagcacat cgccaacctg
     2521 gccggcagcc ccgccatcaa gaagggcatc ctgcagaccg tgaaggtggt ggacgagctg
     2581 gtgaaggtga tgggccgcca caagcccgag aacatcgtga tcgagatggc ccgcgagaac
     2641 cagaccaccc agaagggcca gaagaacagc cgcgagcgca tgaagcgcat cgaggagggc
     2701 atcaaggagc tgggcagcca gatcctgaag gagcaccccg tggagaacac ccagctgcag
     2761 aacgagaagc tgtacctgta ctacctgcag aacggccgcg acatgtacgt ggaccaggag
     2821 ctggacatca accgcctgag cgactacgac gtggaccaca tcgtgcccca gagcttcctg
     2881 aaggacgaca gcatcgacaa caaggtgctg acccgcagcg acaagaaccg cggcaagagc
     2941 gacaacgtgc ccagcgagga ggtggtgaag aagatgaaga actactggcg ccagctgctg
     3001 aacgccaagc tgatcaccca gcgcaagttc gacaacctga ccaaggccga gcgcggcggc
     3061 ctgagcgagc tggacaaggc cggcttcatc aagcgccagc tggtggagac ccgccagatc
     3121 accaagcacg tggcccagat cctggacagc cgcatgaaca ccaagtacga cgagaacgac
     3181 aagctgatcc gcgaggtgaa ggtgatcacc ctgaagagca agctggtgag cgacttccgc
     3241 aaggacttcc agttctacaa ggtgcgcgag atcaacaact accaccacgc ccacgacgcc
     3301 tacctgaacg ccgtggtggg caccgccctg atcaagaagt accccaagct ggagagcgag
     3361 ttcgtgtacg gcgactacaa ggtgtacgac gtgcgcaaga tgatcgccaa gagcgagcag
     3421 gagatcggca aggccaccgc caagtacttc ttctacagca acatcatgaa cttcttcaag
     3481 accgagatca ccctggccaa cggcgagatc cgcaagcgcc ccctgatcga gaccaacggc
     3541 gagaccggcg agatcgtgtg ggacaagggc cgcgacttcg ccaccgtgcg caaggtgctg
     3601 agcatgcccc aggtgaacat cgtgaagaag accgaggtgc agaccggcgg cttcagcaag
     3661 gagagcatcc tgcccaagcg caacagcgac aagctgatcg cccgcaagaa ggactgggac
     3721 cccaagaagt acggcggctt cgacagcccc accgtggcct acagcgtgct ggtggtggcc
     3781 aaggtggaga agggcaagag caagaagctg aagagcgtga aggagctgct gggcatcacc
     3841 atcatggagc gcagcagctt cgagaagaac cccatcgact tcctggaggc caagggctac
     3901 aaggaggtga agaaggacct gatcatcaag ctgcccaagt acagcctgtt cgagctggag
     3961 aacggccgca agcgcatgct ggccagcgcc ggcgagctgc agaagggcaa cgagctggcc
     4021 ctgcccagca agtacgtgaa cttcctgtac ctggccagcc actacgagaa gctgaagggc
     4081 agccccgagg acaacgagca gaagcagctg ttcgtggagc agcacaagca ctacctggac
     4141 gagatcatcg agcagatcag cgagttcagc aagcgcgtga tcctggccga cgccaacctg
     4201 gacaaggtgc tgagcgccta caacaagcac cgcgacaagc ccatccgcga gcaggccgag
     4261 aacatcatcc acctgttcac cctgaccaac ctgggcgccc ccgccgcctt caagtacttc
     4321 gacaccacca tcgaccgcaa gcgctacacc agcaccaagg aggtgctgga cgccaccctg
     4381 atccaccaga gcatcaccgg tctgtacgag acccgcatcg acctgagcca gctgggcggc
     4441 gacggcggct ccggacctcc aaagaaaaag agaaaagtat acccctacga cgtgcccgac
     4501 tacgcctgtt aagcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     4561 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     4621 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     4681 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4741 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4801 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4861 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4921 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4981 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5041 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5101 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5161 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5221 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     5281 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     5341 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     5401 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     5461 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     5521 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     5581 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     5641 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     5701 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     5761 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     5821 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     5881 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     5941 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     6001 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     6061 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     6121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6241 gcggtggttt gtttgccgga tcaagagcca ccaactcttt ttccgaaggt aactggcttc
     6301 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     6361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6841 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6901 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     6961 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     7021 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     7081 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     7141 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     7201 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     7261 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     7321 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     7381 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     7441 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     7501 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     7561 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     7621 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     7681 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     7741 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     7801 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     7861 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     7921 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     7981 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     8041 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     8101 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     8161 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     8221 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     8281 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     8341 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     8401 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     8461 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     8521 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     8581 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     8641 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     8701 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     8761 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     8821 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     8881 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     8941 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     9001 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     9061 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     9121 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     9181 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     9241 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     9301 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     9361 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     9421 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     9481 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     9541 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     9601 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     9661 aat
//
