LOCUS       sequence-309941-       10641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inducible expression of Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10641)
  TITLE     Lian Lab PB plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10641)
  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: 
            "Unpublished"
COMMENT     Sequence Label: PB-iCas9
FEATURES             Location/Qualifiers
     source          1..10641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1051..1365
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1059..1077
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1095..1113
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1130..1148
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1166..1184
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1202..1220
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1237..1255
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1273..1291
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1338..1362
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1382..1391
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1391..1456
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1463..1483
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1508..5608
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1508..5608
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5609..5656
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        5666..6176
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6095..6113
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             6186..6782
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6186..6205)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6682..6702
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6783..6836
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6837..7454,7455..7460,7461..7496,7497..7499,7500..7535,
                     7536..7538,7539..7574,7575..7583)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(7623..7643)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8041..8052)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   8340..8402
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8453..8473)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8453..8471)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8492..8508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8492..8508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8505..8527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8516..8532
                     /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)."
     promoter        complement(join(8540..8546,8547..8564,8565..8570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8585..8606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8723..8740)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8894..9482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8974..8993)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9653..10444,10445..10513))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     10276..10295
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10514..10618)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagccgg gtttattaca gggacagcag
     1021 agatccactt tggccgcgaa tcgatatgtc gagtttactc cctatcagtg atagagaacg
     1081 tatgtcgagt ttactcccta tcagtgatag agaacgatgt cgagtttact ccctatcagt
     1141 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
     1201 ctccctatca gtgatagaga acgtatgtcg agtttatccc tatcagtgat agagaacgta
     1261 tgtcgagttt actccctatc agtgatagag aacgtatgtc gaggtaggcg tgtacggtgg
     1321 gaggcctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag aattggctag
     1381 cgccaccatg gactataagg accacgacgg agactacaag gatcatgata ttgattacaa
     1441 agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
     1501 agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
     1561 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     1621 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     1681 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     1741 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     1801 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     1861 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     1921 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     1981 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     2041 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     2101 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     2161 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     2221 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     2281 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     2341 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     2401 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     2461 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     2521 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     2581 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     2641 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     2701 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     2761 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     2821 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     2881 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     2941 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     3001 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     3061 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     3121 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     3181 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     3241 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     3301 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     3361 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     3421 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     3481 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     3541 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     3601 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     3661 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     3721 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     3781 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     3841 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     3901 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     3961 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     4021 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     4081 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     4141 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     4201 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     4261 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     4321 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     4381 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     4441 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     4501 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     4561 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     4621 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     4681 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     4741 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     4801 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
     4861 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     4921 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     4981 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     5041 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     5101 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     5161 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     5221 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     5281 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     5341 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
     5401 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     5461 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     5521 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     5581 gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca
     5641 ggcaaaaaag aaaaagtaag gatccggggt tggggttgcg ccttttccaa ggcagccctg
     5701 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
     5761 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
     5821 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
     5881 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
     5941 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
     6001 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
     6061 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
     6121 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccagcaat
     6181 tcaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     6241 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     6301 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     6361 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     6421 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     6481 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     6541 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     6601 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     6661 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     6721 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     6781 ccgaaggtag aggttctctc ctcacttgtg gtgatgttga agaaaaccct ggtccaatgt
     6841 ctagactgga caagagcaaa gtcataaacg gagctctgga attactcaat ggtgtcggta
     6901 tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag cctaccctgt
     6961 actggcacgt gaagaacaag cgggccctgc tcgatgccct gccaatcgag atgctggaca
     7021 ggcatcatac ccacttctgc cccctggaag gcgagtcatg gcaagacttt ctgcggaaca
     7081 acgccaagtc ataccgctgt gctctcctct cacatcgcga cggggctaaa gtgcatctcg
     7141 gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc
     7201 agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc cactttacac
     7261 tgggctgcgt attggaggaa caggagcatc aagtagcaaa agaggaaaga gagacaccta
     7321 ccaccgattc tatgccccca cttctgagac aagcaattga gctgttcgac cggcagggag
     7381 ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggag aaacagctaa
     7441 agtgcgaaag cggcgggccg accgacgccc ttgacgattt tgacttagac atgctcccag
     7501 ccgatgccct tgacgacttt gaccttgata tgctgcctgc tgacgctctt gacgattttg
     7561 accttgacat gctccccggg taactaagta aggatcgatc caagatatcg tattcttaac
     7621 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     7681 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     7741 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     7801 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     7861 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     7921 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca
     7981 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     8041 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     8101 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcac
     8161 tagttaaaag ttttgttact ttatagaaga aattttgagt ttttgttttt ttttaataaa
     8221 taaataaaca taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa
     8281 taaaacttaa tatctattca aattaataaa taaacctcga tatacagacc gataaaacac
     8341 atgcgtcaat tttacgcatg attatcttta acgtacgtca caatatgatt atctttctag
     8401 ggttaatcta gtatacgcgt atgcggccgc ttaattaatc cagcttttgt tccctttagt
     8461 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     8521 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     8581 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     8641 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     8701 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     8761 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     8821 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     8881 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     8941 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     9001 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     9061 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     9121 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     9181 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     9241 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     9301 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     9361 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     9421 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     9481 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     9541 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     9601 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     9661 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     9721 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     9781 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     9841 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     9901 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     9961 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
    10021 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
    10081 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
    10141 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
    10201 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
    10261 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
    10321 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
    10381 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
    10441 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
    10501 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
    10561 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
    10621 catttccccg aaaagtgcca c
//
