LOCUS       sequence-334930-       10527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 and GFP upon doxycycline treatment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10527)
  AUTHORS   Ream MW, Randolph LN, Jiang Y, Chang Y, Bao X, Lian XL
  TITLE     Direct programming of human pluripotent stem cells into endothelial 
            progenitors with SOX17 and FGF2.
  JOURNAL   Stem Cell Reports. 2024 Mar 5:S2213-6711(24)00044-4. doi: 
            10.1016/j.stemcr.2024.02.006.
  PUBMED    38518781
REFERENCE   2  (bases 1 to 10527)
  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: "Stem Cell 
            Reports. 2024 Mar 5:S2213-6711(24)00044-4. doi: 
            10.1016/j.stemcr.2024.02.006."
COMMENT     Sequence Label: XLone-Cas9-2A-GFP
FEATURES             Location/Qualifiers
     source          1..10527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..368
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    81..99
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    117..135
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    153..171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    189..207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    225..243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             375..4478
                     /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             375..4478
                     /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             375..4478
                     /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             4491..4511
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4527..4580
                     /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(4581..4583,4584..4586,4587..5300)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4581..4583,4584..4586,4587..5297)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4626..4647)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4887..4906)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5234..5255
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5481..5615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   5891..5925
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5936..5953)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5936..5952)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5945..5967)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6161..6180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6280..6302
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6340..6358)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6426..6530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6531..6599,6600..7391)
                     /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     complement(6749..6768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7562..8150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8051..8070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8304..8321
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8438..8459
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8474..8479,8480..8497,8498..8504)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8512..8528
                     /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)."
     primer_bind     8517..8539
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8536..8552
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8536..8552
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   8578..8640
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    8816..8897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(9014..9412)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(9413..9469)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(9479..10222)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(10223..10246)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      10246..10255
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(10275..10486)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag
       61 agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc
      121 tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta
      181 cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag
      241 agaacgtata agctttgctt atgtaaacca gggcgcctat aaaagagtgc tgattttttg
      301 agtaaacttc aattccacaa cacttttgtc ttataccaac tttccgtacc acttcctacc
      361 ctcgtaaagg taccatggac aagaagtact ccattgggct cgatatcggc acaaacagcg
      421 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
      481 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
      541 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
      601 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
      661 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
      721 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
      781 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
      841 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
      901 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
      961 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
     1021 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
     1081 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
     1141 tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     1201 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     1261 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     1321 cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga
     1381 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     1441 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     1501 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     1561 aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc
     1621 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     1681 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     1741 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
     1801 ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     1861 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     1921 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     1981 agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga
     2041 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg
     2101 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
     2161 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
     2221 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
     2281 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat
     2341 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
     2401 gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt
     2461 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
     2521 agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
     2581 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
     2641 ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga
     2701 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
     2761 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
     2821 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
     2881 acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
     2941 tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg
     3001 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
     3061 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct
     3121 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
     3181 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
     3241 ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga
     3301 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
     3361 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
     3421 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
     3481 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
     3541 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
     3601 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
     3661 aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca
     3721 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
     3781 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
     3841 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
     3901 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
     3961 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
     4021 gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
     4081 tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc
     4141 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
     4201 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
     4261 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
     4321 ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt
     4381 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
     4441 atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga
     4501 agaggaaggt gaggtccggc ggcggagagg gcagaggaag tcttctaaca tgcggtgacg
     4561 tggaggagaa tcccggccca atggtgagca agggcgagga gctgttcacc ggggtggtgc
     4621 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     4681 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     4741 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     4801 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     4861 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     4921 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     4981 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     5041 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     5101 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     5161 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     5221 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     5281 tggacgagct gtacaagtaa actagtagac cacctcccct gcgagctaag ctggacagcc
     5341 aatgacgggt aagagagtga catttttcac taacctaaga caggagggcc gtcagagcta
     5401 ctgcctaatc caaagacggg taaaagtgat aaaaatgtat cactccaacc taagacaggc
     5461 gcagcttccg agggatttga gatccagaca tgataagata cattgatgag tttggacaaa
     5521 ccaaaactag aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct
     5581 tatttgtaac cattataagc tgcaataaac aagtttgata tctataacaa gaaaatatat
     5641 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     5701 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     5761 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     5821 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     5881 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg
     5941 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     6001 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     6061 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     6121 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     6181 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6241 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     6301 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     6361 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     6421 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6481 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6541 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6601 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6661 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6721 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6781 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6841 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6901 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6961 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     7021 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7081 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7141 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7201 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     7261 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7321 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7381 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7441 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7501 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7561 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7621 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7681 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7741 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7801 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7861 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7921 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7981 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8041 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8101 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8161 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     8221 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     8281 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     8341 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     8401 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     8461 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     8521 gataacaatt tcacacagga aacagctatg accatgatta cgccaaggtc gacttaaccc
     8581 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
     8641 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
     8701 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
     8761 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttagcagt
     8821 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     8881 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     8941 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
     9001 atcctctgga gatttagccc tcccacacat aaccagaggg cagcaattca cgaatcccaa
     9061 ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcagatcga
     9121 gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg
     9181 cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt
     9241 ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc
     9301 tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag
     9361 ccgttgctct ttcaatgagg gtggattctt cttgagacaa aggcttggcc atggggccgg
     9421 ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc
     9481 cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa
     9541 agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg
     9601 gcccgccgct ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga
     9661 aaaggaaggc aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg
     9721 ggggcataga atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt
     9781 cctccaatac gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct
     9841 ccagggagaa gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt
     9901 tctctgttgg gcgggtgccg agatgcactt tagccccgtc gcgatgtgag aggagagcac
     9961 agcggtatga cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc
    10021 aggagtgggt atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct
    10081 tgttcttcac gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc
    10141 ttgtcgtcag gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt
    10201 tgctcttgtc cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc
    10261 ccgcgtcacg acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg
    10321 actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg
    10381 acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc
    10441 cctcccccca acttctcggg gactgtgggc gatgtgcgct ctgcccactg acgggcaccg
    10501 gagccactcg agtggaatta tcacctc
//
