LOCUS       sequence-466941-       11510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  the TLCV2 plasmid with Tol2 recombination sites for integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11510)
  AUTHORS   Moses E, Franek R, Harel I
  TITLE     A scalable and tunable platform for functional interrogation of 
            peptide hormones in fish.
  JOURNAL   eLife. 2023 Oct 24;12:e85960. doi: 10.7554/eLife.85960.
  PUBMED    37872843
REFERENCE   2  (bases 1 to 11510)
  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: "eLife."; 
            date: "2023-10-24"; volume: "12:e85960. doi"; pages: " 
            10.7554/eLife.85960"
COMMENT     Sequence Label: Tol2-TLCV2
FEATURES             Location/Qualifiers
     source          1..11510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     1061..1077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1097..1220
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1246..1561
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1254..1272
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1290..1308
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1325..1343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1361..1379
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1397..1415
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1433..1451
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1469..1487
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1534..1558
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1587..1596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1593..5696
                     /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             1593..5696
                     /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             5697..5744
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5745..5768
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5778..5831
                     /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(5832..5834,5835..6548)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5832..5834,5835..6545)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6135..6154)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6482..6503
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        6559..6770
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6792..7388
                     /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(6792..6811)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7288..7308
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             7398..7454
                     /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             join(7455..8072,8073..8078,8079..8114,8115..8117,8118..8153,
                     8154..8156,8157..8192,8193..8201)
                     /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(8219..8236)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8225..8432
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    8467..8601
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8491..8510)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8545..8564
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(9040..9227,9228..9239)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(9273..9289)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9273..9289)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(9326..9346)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9326..9344)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9365..9381)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9378..9400)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9389..9405
                     /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(9413..9419,9420..9437,9438..9443))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9458..9479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9596..9613)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9767..10355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9847..9866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10526..11317,11318..11386))
                     /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     11149..11168
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11387..11491)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 atgctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatg ggccctcgag caggaaacag ctatgaccat
     1081 gattacgcca agctatcaac tttgtataga aaagttgaac gagaaacgta aaatgatata
     1141 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     1201 caacatatcc agtcactatg gggtaccgcc gcgaatcgat atgtcgagtt tactccctat
     1261 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gatgtcgagt
     1321 ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
     1381 cgtatgtcga gtttantccc tatcagtgat agagaacgta tgtcgagttt actccctatc
     1441 agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagg
     1501 taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc
     1561 ctggagaata ccggttctag agcgctgcca ccatggacaa gaagtacagc atcggcctgg
     1621 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     1681 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     1741 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     1801 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1861 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1921 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1981 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     2041 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     2101 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     2161 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     2221 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     2281 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     2341 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     2401 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     2461 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     2521 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     2581 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     2641 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     2701 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2761 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2821 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2881 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2941 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     3001 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     3061 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     3121 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     3181 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     3241 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     3301 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     3361 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     3421 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     3481 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     3541 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     3601 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     3661 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3721 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3781 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3841 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3901 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3961 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     4021 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     4081 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     4141 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     4201 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     4261 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     4321 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     4381 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     4441 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     4501 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     4561 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     4621 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     4681 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4741 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     4801 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     4861 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4921 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     4981 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     5041 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     5101 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     5161 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     5221 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     5281 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     5341 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     5401 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     5461 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     5521 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     5581 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     5641 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaagc
     5701 gacctgccgc cacaaagaag gctggacagg ctaagaagaa gaaagattac aaagacgatg
     5761 acgataaggg atccggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     5821 atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     5881 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     5941 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     6001 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     6061 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     6121 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     6181 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     6241 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     6301 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     6361 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     6421 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     6481 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     6541 acaagtaagc tagcatccgg gcagagcgca catcgcccac agtccccgag aagttggggg
     6601 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     6661 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     6721 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag actgcgatcg
     6781 caatgtacag tatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     6841 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     6901 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     6961 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     7021 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     7081 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     7141 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca
     7201 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     7261 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     7321 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     7381 ccggtgccgg ttccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag
     7441 agaatcctgg accgatgtct agactggaca agagcaaagt cataaacgga gctctggaat
     7501 tactcaatgg tgtcggtatc gaaggcctga cgacaaggaa actcgctcaa aagctgggag
     7561 ttgagcagcc taccctgtac tggcacgtga agaacaagcg ggccctgctc gatgccctgc
     7621 caatcgagat gctggacagg catcataccc acttctgccc cctggaaggc gagtcatggc
     7681 aagactttct gcggaacaac gccaagtcat accgctgtgc tctcctctca catcgcgacg
     7741 gggctaaagt gcatctcggc acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc
     7801 agctcgcgtt cctgtgtcag caaggcttct ccctggagaa cgcactgtac gctctgtccg
     7861 ccgtgggcca ctttacactg ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag
     7921 aggaaagaga gacacctacc accgattcta tgcccccact tctgagacaa gcaattgagc
     7981 tgttcgaccg gcagggagcc gaacctgcct tccttttcgg cctggaacta atcatatgtg
     8041 gcctggagaa acagctaaag tgcgaaagcg gcgggccgac cgacgccctt gacgattttg
     8101 acttagacat gctcccagcc gatgcccttg acgactttga ccttgatatg ctgcctgctg
     8161 acgctcttga cgattttgac cttgacatgc tccccgggta aagcggcccg ctgatcagcc
     8221 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     8281 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     8341 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     8401 gattgggaag acaatagcag gcatgctggg gagagatcgc cgccaccgcg gtgtaccatc
     8461 gatgatgatc cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     8521 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     8581 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     8641 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat
     8701 tatgatcctc tagatcagat ctgcgaagat acggccacgg gtgctcttga tcctgtggct
     8761 gattttggac tgtgctgctc gcagctgctg atgaatcaca tacttcctcc attttcttcc
     8821 actgattgac tgttataatt tccctaattt ccaggtcaag gtgctgtgca ttgtggtaat
     8881 agatgtgaca tgacgtcact tccaaaggac caatgaacat gtctgaccaa tttcatataa
     8941 tgtgaaaacg attttcatag gcagaataaa taacatttaa attaaactgg gcatcagcgc
     9001 aattcaattg gtttggtaat agcaagggaa aatagaatga agtgatctcc aaaaaataag
     9061 tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat
     9121 gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc
     9181 caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt acacctctgg
     9241 ttcttgaccc cctaccttca gcaagcccag cagatccact agttctagag cggccgccac
     9301 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     9361 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     9421 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     9481 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     9541 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     9601 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     9661 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     9721 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     9781 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     9841 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     9901 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     9961 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
    10021 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
    10081 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
    10141 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
    10201 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
    10261 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
    10321 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
    10381 ggtctgacgc tcagtggaac gaaaactcac gttaaggcat tttggtcatg agattatcaa
    10441 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
    10501 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
    10561 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
    10621 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
    10681 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
    10741 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
    10801 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
    10861 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
    10921 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
    10981 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
    11041 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
    11101 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
    11161 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
    11221 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
    11281 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
    11341 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
    11401 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
    11461 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
//
