LOCUS       sequence-442802-       13905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent Cas9-GFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13905)
  AUTHORS   Hans S, Zoller D, Hammer J, Stucke J, Spiess S, Kesavan G, Kroehne 
            V, Eguiguren JS, Ezhkova D, Petzold A, Dahl A, Brand M
  TITLE     Cre-Controlled CRISPR mutagenesis provides fast and easy conditional
            gene inactivation in zebrafish.
  JOURNAL   Nat Commun. 2021 Feb 18;12(1):1125. doi: 10.1038/s41467-021-21427-6.
  PUBMED    33602923
REFERENCE   2  (bases 1 to 13905)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-02-18"; volume: "12(1):1125. doi"; pages: " 
            10.1038/s41467-021-21427-6"
COMMENT     Sequence Label: pTol2 hsp70l loxP-DsRed-loxP-nCas9n-GFP
FEATURES             Location/Qualifiers
     source          1..13905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(2503..2514,2515..2677)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    complement(4497..4530)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4537..4546
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4543..5220
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(4723..4743)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     5135..5158
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5341..5462
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5716..5749)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             5768..5788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5795..9895
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             9808..9816
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             9911..9931
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(9932..9934,9935..10648)
                     /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(9932..9934,9935..10645)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9974..9995)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(10235..10254)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10582..10603
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    10771..10852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(11435..11622,11623..11634)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(11668..11684)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11668..11684)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(11721..11741)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(11721..11739)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11760..11776)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11760..11776)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11773..11795)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11784..11800
                     /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(11808..11814,11815..11832,11833..11838))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11853..11874
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11991..12008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12162..12750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12242..12261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12921..13712,13713..13781))
                     /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     13544..13563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13782..13886)
                     /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 tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccaaatagta ggaattaccc acctgtacaa gtgctgaaaa cttggatgaa taagcccgtt
      721 tgccattttc tactgctatt ttaaatcttt tctgtatttg tctgcatttg tctttaccct
      781 tgaataaatg ttttagttgt tttttttcaa ttatgactgt gtttaacaga cattattaca
      841 ataatatgta atagtagtac acactattat tatgtaatag tacttgttga ctgtatttga
      901 gaactggacc gagtgagtgt tacgtcaccc attcaaaatg acttacttct ggctccaaca
      961 aaatgaagtt aattcagttg ccatttttca ctgtatggac atcgccgtgt tggagctaga
     1021 cgttgccaat aagcaagaaa gagccgagat gcgtcgagtc tgagtcaccg ttcctatggc
     1081 aacccctcta accaatcaga agtaagcttg ttggaagtcc acagcctacc acttgaaagc
     1141 gggctgcaca aaatctgtca aacgttttga acgttggatg tgagagcaca tacttttatt
     1201 aaggcatctg gttggtcagt ttataatatc aacaacttgg gctacagaaa agaaaagtta
     1261 ttacagaaat tatgattaac aagtacatgt taaataaaga ttttaatatg aatgccacca
     1321 ctggagcatt catgccattt ggagcttctt cctgtttgga tcactagaag gaggaggtca
     1381 ctcattacag ttctcatata cagtcgttgg ttggttggtt ggttggtaga ttgattgatt
     1441 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt
     1501 gattggtagt caaaataaga aataatttcc acagattcat tacagaaatg attaaatgca
     1561 tacataaaaa actggggggg gggggggggg atacaacaac acacttaagt cacatttgcc
     1621 tacgtaaaga aaagtaaaga aaatcaatag ctatatttta catctccttt ttttgctgtc
     1681 ttttaattag ccttgttttg ctgttatctt gattgaaact gtaacttctt cacctgcttc
     1741 ttttctttgt aggttttgcc agcccagaaa caggcatggc tgtgcaaggc cagagcacca
     1801 tgcacaatgc gctgcatgtc ttcatgaacg gctcaatgtc ctcagtccag ggctcagcca
     1861 acgaccccat cttcctcctt caccatgctt tcattgacag gtaacaaaca cgtcatgaca
     1921 ttagactgca cagtttttga caaagttcat acaatctgtt gtttatagct gctacaatta
     1981 gtgaagtttg tgaatgtact tggatgagca gcgaaagatc aattgagatc aattgttaga
     2041 gtttggttgc cctgcagagc aaagaacaaa aaataatctg gtggctttac tgcgtgaggt
     2101 tattattggt ggaatagaaa cacaaaacat aattgcattt atttgtttaa ttttttatct
     2161 tatcttaact ttcatcttgc atatttgttt cttacatcat ttctagcatc tttgagcgct
     2221 ggctaagaac tcatcagcct ccccggtcca tctacccacg taccaatgca ccaattggcc
     2281 acaatgacgg ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc
     2341 tgtccaacaa tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa
     2401 caccagaaat gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca
     2461 ctctgttcca cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac
     2521 tcaaaaattt tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag
     2581 taaaagtatc tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg
     2641 tacttgagta ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta
     2701 agcttttaat ctcaaaaaac attaaatgaa atgcatacaa ggttttatcc tgctttagaa
     2761 ctgtttgtat ttaattatca aactataaga cagacaatct aatgccagta cacgctactc
     2821 aaagttgtaa aacctcagat ttaacttcag tagaagctga ttctcaaaat tgttagtgtc
     2881 aagcctagct cttttggggc tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc
     2941 agccgatgcg ggaagaggtg tattagtctt gatagagagg ctgcaaatag caggaaacgt
     3001 gagcagagac tccctggtgt ctgaaacaca ggccagatgg gcccttcagg ggtgtcgctt
     3061 ggttatttcc aaaaatcaaa ttaattttat taaactatta gaacgagcat gttttgtcta
     3121 tatgctacag aagataaaaa ataataggag ttaacagtta taaaacaaca cactttgttt
     3181 ctattgattg ttgaccacac tggggtctca ttaagttaga ttaaagacac actaactggg
     3241 tcaaaagcag cagattgatt tcatagcacc agggtaaact ttctaacact tttacggcaa
     3301 tcatatacat taaaattaaa tacagaccac gactgaacaa ggaggatgat ctccaatatt
     3361 aaacaaagag acttgtgcct atttctctga gggtaaacat gacctctcaa gttagcaagt
     3421 tgtttttaac actacaaaaa tagttaagac tgcaatccca gaataaagta ttggttttaa
     3481 ccaatcaata tagtacagta aacatccatt tgttttgttg aaacgttaaa caaatctgac
     3541 caaagctatt agcttatata aaacaggttt gccttctatg tagctgaaaa caccacaggc
     3601 ccgattttgc tactgtgtaa aacatttcag caagattttt ttattgcatt tttttttact
     3661 gaatcgttca aacattttat cattttagtt tgttcattca ttgcaactgg aaaaacaaca
     3721 catcacacaa ccgcacatat ttcagcaata agtacaataa aacactcaaa taaaaaaaac
     3781 attttaaatc tctttgtatt tttgaccgct gtttcgcgta atttcacggt aaaactctgg
     3841 aaatctccac tacattcctc tcagcggctc ctctcaatga cagctgaaga agtgacgcgg
     3901 ctgcctgctg tgttttgatt ggtcgaattc actggaggct tccagaacag tgtagagtct
     3961 gaacgggtgc gcgctctgct gtatttaaag ggcgaaagag agaccgcaga gaaactcaac
     4021 cgaagagaag cgacttgaca aagaagaaaa gagcagcctg acaggacttt ttccccgacg
     4081 aggtgtttat tcgctctatt taagaatcta ctgtaaggta agtctcaata tattgtactc
     4141 tattggctaa tcagaattat atagagatta tatgtactta atgtcaaaaa atcaactttg
     4201 tatatgtaat ctttttacat gtggactgcc tatgttcatc ttattttagg tctactagaa
     4261 aattatattt cccgttttca caataaggat ttttaaaaaa agcaatgaac agacgggcat
     4321 ttactttatg ttgctgacat tattttatat gagcataata accataaata ctagcaaatg
     4381 tcctaaatga atttgtgtta atgttgtcta caaaagaaaa ttagcgtttt acttgtacaa
     4441 ctaataataa cttggttatt aagagaattt cacttgttga ctagaaaagg ccggccataa
     4501 cttcgtatag catacattat acgaagttat ccggtcgcca ccatggcctc ctccgagaac
     4561 gtcatcaccg agttcatgcg cttcaaggtg cgcatggagg gcaccgtgaa cggccacgag
     4621 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gccacaacac cgtgaagctg
     4681 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag
     4741 tacggctcca aggtgtacgt gaagcacccc gccgacatcc ccgactacaa gaagctgtcc
     4801 ttccccgagg gcttcaagtg ggagcgcgtg atgaactttg aggacggcgg cgtggcgacc
     4861 gtgacccagg actcctccct gcaggacggc tgcttcatct acaaggtgaa gttcatcggc
     4921 gtgaacttcc cctccgacgg ccccgtgatg cagaagaaga ccatgggctg ggaggcctcc
     4981 accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg agacccacaa ggccctgaag
     5041 ctgaaggacg gcggccacta cctggtggag ttcaagtcca tctacatggc caagaagccc
     5101 gtgcagctgc ccggctacta ctacgtggac gccaagctgg acatcacctc ccacaacgag
     5161 gactacacca tcgtggagca gtacgagcgc accgagggcc gccaccacct gttcctgtag
     5221 cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     5281 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     5341 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5401 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5461 taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5521 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5581 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5641 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5701 atcaccctaa tcaagataac ttcgtatagc atacattata cgaagttatc ccgtcatggc
     5761 ttctccacct aagaagaaga gaaaggtggg aagcatggat aagaagtata gcatcggcct
     5821 ggatattgga actaactccg tgggttgggc agtgattaca gacgactaca aggtccctag
     5881 caagaaattt aaggtgctgg gtaacaccga caggcacagc atcaagaaaa atctgattgg
     5941 agccctgctg ttcggttctg gagagactgc cgaagcaaca cgcctgaaaa gaacagcaag
     6001 aaggcgctat accagaagga agaatagaat ctgttacctg caggagattt tctctaacga
     6061 aatggctaag gtggacgatt cattctttca taggctggag gaaagtttcc tggtcgagga
     6121 agataagaaa cacgagcgcc atcctatctt tggaaacatt gtggacgagg tcgcctatca
     6181 cgaaaaatac ccaaccatct atcatctgcg caagaaactg gctgactcta ctgataaagc
     6241 cgacctgaga ctgatctatc tggctctggc ccacatgatt aagttcaggg gtcattttct
     6301 gatcgagggc gatctgaacc ccgacaattc cgatgtggac aagctgttca tccagctggt
     6361 ccagatttac aatcagctgt ttgaggaaaa ccctattaat gcttccagag tggacgcaaa
     6421 agctatcctg tcagccaggc tgtccaagtc acgcagactg gagaacctga ttgcacagct
     6481 gcccggagaa aagaggaacg gtctgtttgg aaatctgatc gctctgagtc tgggcctgac
     6541 tcctaacttc aaaagcaatt ttgatctggc tgaggacgcc aaactgcagc tgtcaaagga
     6601 cacatatgac gatgacctgg ataacctgct ggcacagatc ggagatcagt acgctgacct
     6661 gttcctggct gccaaaaatc tgtccgacgc aatcctgctg tcagatattc tgagagtgaa
     6721 cagcgagatt acaaaagctc ctctgagtgc cagcatgatc aagagatatg acgagcacca
     6781 tcaggatctg accctgctga aggctctggt caggcagcag ctgccagaga agtacaagga
     6841 aattttcttt gatcagtcca agaacggcta cgccggttat atcgacggag gcgcatcaca
     6901 ggaggaattc tacaagttta tcaaacctat tctggagaag atggacggaa ctgaggaact
     6961 gctggtgaaa ctgaatagag aggacctgct gaggaagcag cgcacatttg ataacggttc
     7021 catcccacac cagattcatc tgggagagct gcacgctatc ctgaggcgcc aggaagactt
     7081 ctaccccttt ctgaaagata accgcgagaa gatcgaaaaa attctgacct tcagaatccc
     7141 ttactatgtg ggtccactgg ctcgcggaaa cagcagattt gcctggatga ctcgcaaatc
     7201 cgaggaaacc attactcctt ggaacttcga ggaagtggtc gataagggcg cctctgcaca
     7261 gtccttcatc gagagaatga ctaattttga caaaaacctg cccaatgaga aagtgctgcc
     7321 taagcactcc ctgctgtacg agtatttcac tgtctataac gaactgacaa aggtgaaata
     7381 cgtcaccgag ggcatgagaa agccagcctt cctgtcagga gagcagaaga aagcaatcgt
     7441 ggatctgctg tttaaaacca acaggaaagt gactgtcaag cagctgaagg aggactactt
     7501 caagaaaatt gaatgcttcg attccgtgga gatcagcgga gtcgaagaca gatttaacgc
     7561 aagcctgggc gcttaccacg atctgctgaa gatcattaag gataaagact tcctggacaa
     7621 cgaggaaaat gaggatatcc tggaagacat tgtgctgaca ctgaccctgt ttgaggacag
     7681 aggaatgatc gaggaaagac tgaaaaccta tgctcatctg ttcgatgaca aggtgatgaa
     7741 acagctgaag agaaggcgct acactggctg gggtagactg agcaggaagc tgatcaacgg
     7801 cattagggat aaacagtcag gaaagacaat cctggacttt ctgaaaagtg atggcttcgc
     7861 caaccgcaat tttatgcagc tgattcacga tgacagtctg accttcaaag aggacatcca
     7921 gaaggctcag gtgtctggac agggccactc cctgcatgag cagattgcaa acctggctgg
     7981 aagcccagcc atcaagaaag gcattctgca gacagtgaaa atcgtcgatg agctggtgaa
     8041 agtcatgggc cataagcccg aaaacatcgt gattgagatg gctcgcgaaa atcagacaac
     8101 ccagaagggt cagaagaaca gtagagagag gatgaaaaga atcgaggaag gcattaagga
     8161 gctgggtagc cagatcctga aagagcaccc agtggaaaac acacagctgc agaatgagaa
     8221 gctgtatctg tactatctgc agaatggaag agatatgtac gtggaccagg agctggatat
     8281 taacaggctg tctgattacg acgtggatca tatcgtcccc cagagtttca tcaaagatga
     8341 cagcattgac aacaaggtgc tgaccaggtc cgacaaaaac agaggaaaat cagataatgt
     8401 ccctagtgag gaagtggtca agaaaatgaa gaactactgg agacagctgc tgaatgccaa
     8461 actgatcact cagaggaagt ttgataacct gacaaaagca gagcgcggtg gactgtcaga
     8521 actggacaaa gctggattca tcaagaggca gctggtggaa acacgccaga tcactaaaca
     8581 cgtcgcacag attctggata gtcgcatgaa cacaaagtac gatgagaatg acaaactgat
     8641 cagagaagtg aaggtcatta ccctgaagag taaactggtc agcgacttta ggaaagattt
     8701 ccagttttat aaggtccgcg agattaacaa ttatcaccat gcccatgacg catacctgaa
     8761 cgccgtggtc ggtaccgcac tgatcaagaa atacccaaaa ctggagagcg aattcgtgta
     8821 cggagactat aaggtgtacg atgtcagaaa aatgatcgcc aagtccgagc aggaaattgg
     8881 aaaagctact gccaagtatt tcttttactc aaacatcatg aatttcttta agacagagat
     8941 caccctggcc aatggagaaa tccgcaaaag gcccctgatt gagacaaacg gagagacagg
     9001 cgaaatcgtg tgggacaaag gcagagattt tgcaaccgtg aggaaggtcc tgagcatgcc
     9061 tcaagtgaat atcgtcaaga aaactgaggt gcagacaggc ggtttctcaa aagaaagtat
     9121 tctgccaaaa cgcaactctg ataagctgat cgctagaaag aaagactggg accctaagaa
     9181 gtatggaggc tttgactctc ccactgtggc atactccgtc ctggtggtcg ctaaggtgga
     9241 gaagggcaaa agcaagaaac tgaaatctgt caaggagctg ctgggtatca caattatgga
     9301 gagaagctct ttcgagaaga acccaatcga ttttctggag gccaaaggtt ataaggaagt
     9361 gaagaaagac ctgatcatta aactgcccaa gtacagtctg tttgagctgg aaaacggcag
     9421 gaaacgcatg ctggcaagcg ctggagagct gcagaaaggc aatgaactgg ccctgccttc
     9481 taagtacgtg aacttcctgt atctggcaag ccactacgag aagctgaaag gatctccaga
     9541 ggataacgaa cagaaacagc tgtttgtgga gcagcacaag cattatctgg acgagatcat
     9601 tgaacagatt agcgagttct ctaaaagagt gatcctggcc gacgcaaatc tggataaggt
     9661 cctgtctgct tacaacaaac acagagataa gcccatcagg gagcaggccg aaaatatcat
     9721 tcatctgttc actctgacaa acctgggcgc acctgcagct ttcaagtact tcgacactac
     9781 aatcgataga aagaggtaca cctccactaa ggaggtgctg gacgctacac tgatccatca
     9841 gagtattacc ggcctgtacg aaacaaggat tgacctgtct cagctgggtg gcgactcacc
     9901 ggtgagatct cctaagaaga agagaaaggt ggtgagcaag ggcgaggagc tgttcaccgg
     9961 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
    10021 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
    10081 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
    10141 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
    10201 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
    10261 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
    10321 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
    10381 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
    10441 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
    10501 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
    10561 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
    10621 tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat
    10681 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
    10741 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
    10801 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
    10861 tgtggtttgt ccaaactcat caatgtatcg ctagcatata tggcgcgcca tcaacgtcaa
    10921 aacatgcaca cccaaaaaaa aaaaaaaaaa aaaaaaaact atcagtaaaa caaaatactt
    10981 aatgtactta cctcaatatg cttccttagg tttgatggcg aacttttgaa ggccgatatt
    11041 tctttattaa gcgggagaca gaggacacat ttcatcttga atgaatcttt atttacacca
    11101 cttaaagaaa agaattcgcg aagatacggc cacgggtgct cttgatcctg tggctgattt
    11161 tggactgtgc tgctcgcagc tgctgatgaa tcacatactt cctccatttt cttccactga
    11221 ttgactgtta taatttccct aatttccagg tcaaggtgct gtgcattgtg gtaatagatg
    11281 tgacatgacg tcacttccaa aggaccaatg aacatgtctg accaatttca tataatgtga
    11341 aaacgatttt cataggcaga ataaataaca tttaaattaa actgggcatc agcgcaattc
    11401 aattggtttg gtaatagcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt
    11461 tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat
    11521 taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa
    11581 ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt
    11641 gaccccctac cttcagcaag cccagcagat ccactagttc tagagcggcc gccaccgcgg
    11701 tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
    11761 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
    11821 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
    11881 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
    11941 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
    12001 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
    12061 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
    12121 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
    12181 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
    12241 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
    12301 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
    12361 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
    12421 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
    12481 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
    12541 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
    12601 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
    12661 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
    12721 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
    12781 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
    12841 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
    12901 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
    12961 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
    13021 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
    13081 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
    13141 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
    13201 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
    13261 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
    13321 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
    13381 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
    13441 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
    13501 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
    13561 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
    13621 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
    13681 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
    13741 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
    13801 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
    13861 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtg
//
