LOCUS       sequence-458642-       13926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double inducible expression of Cas12a+ with Gal4 and Flp..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13926)
  AUTHORS   Port F, Buhmann MA, Zhou J, Stricker M, Vaughan-Brown A, Michalsen 
            A-C, Roßmanith E, Poltl A, Großkurth L, Huber J, Menendez Kury LB, 
            Weberbauer B, Hubl M, Heigwer F, Boutros M
  TITLE     Enhanced in vivo gene knockout with undetectable off-targets using 
            multiplexed Cas12a sgRNAs
  JOURNAL   bioRxiv 2024.11.26.625385
REFERENCE   2  (bases 1 to 13926)
  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: "bioRxiv 
            2024.11.26.625385"
COMMENT     Sequence Label: UAST-FRT-GFP-FRT-uMCas12a+nls1x
FEATURES             Location/Qualifiers
     source          1..13926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(193..213)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          343..408
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(490..570)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(577..624)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(625..4308)
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFRNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VKH"
     protein_bind    4436..4469
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    4487..4621
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4893..4913)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5043..5108
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(join(5216..5926,5927..5929,5930..5932))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5258..5279)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5607..5626
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    5937..5970
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(5999..6237)
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     complement(6031..6052)
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     protein_bind    6256..6350
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     protein_bind    complement(6383..6416)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(7191..7805,7876..8007,8211..8526,8588..9242,
                     9317..9590,9992..10063))
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     primer_bind     10515..10533
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     primer_bind     complement(10566..10585)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10567..10585)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10592..10609)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10592..10608)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10601..10623)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10750..11205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10837..10856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11047..11068
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11231..11335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11336..11404,11405..12196)
                     /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(11554..11573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12367..12955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12856..12875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13109..13126
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13243..13264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13279..13284,13285..13302,13303..13309)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13317..13333
                     /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     13322..13344
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13341..13357
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13341..13357
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        13378..13396
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    13615..13684
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     polyA_signal    13713..13847
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 acaaatgtgg tatggctgat tatgatctct agtcaaggca ctatacatca aatatttttc
       61 cataattttc ttgtatagca gtgcagcttt ttcctttgtg gtgtaaatag caaagcaagc
      121 aagagttcta ttactaaaca cagcatgact caaaaaactt agcaattctg aaggaaagtc
      181 cttggggtct tctacctttc tcttcttttt tggaggagta gaatgttgag agtcagcagt
      241 agcctcatca tcactagatg gcatttcttc tgagcaaaac aggttttcct cattaaaggc
      301 attccaccac tgctcccatt catcagttcc ataggttgga atctaaaata cacaaacaat
      361 tagaatcagt agtttaacac attatacact taaaaatttt atatttacct tagagcttta
      421 aatctctgta ggtagtttgt ccaattatgt cacaccacag aagtaaggtt ccttcacaaa
      481 gatcctttag gcatagtcgg ggacatcata tgggtatgca taatcaggca cgtcgtaggg
      541 ataagcgtaa tctggaacat cgtatgggta ggatcccttt ttcttttttg cctggccggc
      601 ctttttcgtg gccgccggcc ttttgtgctt cacgctggtc tgggcgtact ccagccactc
      661 cttgttagag atggcgatct tcaccttatc cagcttctcg tcctcggcct tcttgaactg
      721 gccgatggcc cacagcacct ttctggcgat gttataggcg ccattggcgt cggcgttctt
      781 tggcaggatg gcattctcct gggcctcata gttccggcta tcgtagaaga tgccgtcgga
      841 gttcttcaca gggctgatca gaaaatccac gtcggtgcgg cctgtgatgc tgttccgcat
      901 ctgcagcatc aggctcatca gggccataaa gctagagtag aaggccttgt cggactgctc
      961 gcacagcagg gctctgatat cgccctgctg ataattgatg ccgtacttgt tgaacagctc
     1021 cttataggcg ctggtcaggc acacctcctc ccagtcgaac acgttgttct tcttaggatt
     1081 ccggaagatt ctgatccggt tgccgtagga gtacagcttc cacttcttga tgtaatcggc
     1141 gtctgtgcga gagaagttct tatagtccag ggcaaactcg aacagatcct cctcgggcac
     1201 gtacatgatc ctgtcaaagg agctgatgaa cttcttggaa tcggcgatgc tggtatactt
     1261 ggttttcagc aggttcacaa agccggtaga tggatcgatc ttggatgtca gccaggcagg
     1321 gatgtaaaag atgaagccgt tctgggtaga catggactta aagctctcga acttattggt
     1381 gatctgatag cccttcaggg cgccgcctgt tgcacaagga ttagacttct tgtccaccat
     1441 gtagttcagc ttatcgatca gcatcttctc gaacttctga tacacctgct tctccacctt
     1501 cacgcggcta ttcttaaagc cagagttcag gtcctccagg gcgatcacgg catcgtactt
     1561 ctccaccagc tcgcagatct tgtgcaccac ctgagagata tagccggcct tcagctcctt
     1621 gatattctcg atggaggtcc agttctggcg ggcctcgaac ctctccttct ccttcttgtc
     1681 cagcagagag tggtaatctg tcttgatcct gatgccgttg aagttgttga tgatctcgtt
     1741 cagggaatac tgctccacga tgttgccctt gccgtccacc accacgatat acagcagatt
     1801 gcgctcgccc ctatcgatgc cgatcacata ggggttatcg tcgtgcttca gcagcacgcg
     1861 cacctctgta ttgatcttga agatgttctt ggggcactta ttgatggcga ttgggatgtg
     1921 cagctcgtac tggtcctcag aaaacctctt atccttatac acgtcgtagg acagggttgt
     1981 ggttttcttg ggattatctg gattcttgtt ggcgataggg gagttggctg ggtgcaccac
     2041 cagctcctcc ttcttcaggg aggcgcgcct catgaacagc tctgctcctc cgctcagcct
     2101 gatctgtccg tgattgttct cgtcaaacag cagcttgaag tacatggtgt gcagattggg
     2161 tgtgccgtga gacttatcgg aaaagtcctt gttatagatc tggaacatat acagcttgcc
     2221 ctcctccacc agcttatcca cctccttctt gctggcagac tcgaagctca ccttatagcc
     2281 ctgctcctcc acctctctgt aaaagccggc gatgtcctta tacttctctg tctcagaaaa
     2341 gttgaaatcg taggcattgg accactttgg ataccgggag atgctatcct taaagaagtc
     2401 gatcagcttg tgacagtcat tcaggttaaa catatcgccc ttcttgaatg tgccattctt
     2461 gtagatcttc tggatgtcct cgctggggtt atagtaggcc atccacttct tagaaaagaa
     2521 cacctttggc agcatcttat tagggccggg cagcagctta tagttgatct tctcgtaatt
     2581 gccgttcaca tcgtccttgt cgatcttctg caggcacttg gcgtacttct tatccatgat
     2641 ggccagatag tacttggagc cgtatctcag gatggtggcc cgatagtctg tctccttatc
     2701 cttgtcccag ccgcccatga actgagggtt ctgaaaatac agcttgaact tatccttaga
     2761 gtagggcttc tgggtcacat aattgcggat ggcatcgtag atgtggtcca ccttcagcag
     2821 gatgtcgtag gccagcacaa aatcgccata gaaggactcg tccctgtttg tctccttgcc
     2881 ctcgccaaag aaggccttga tgtaattctc gaagctcttc acagaatcca gcaggtcctt
     2941 catgatggcc accacggcgt cgttcttctt caggctcttc tccagcacaa aatcggcgtc
     3001 gaacagcttc tcagaggagc catacacctt gtagatctca tccaccttct ggatgatgat
     3061 ctccttcagc ttctccacca cagacagatc ggcgtcggcg tactcctgca gctgctccag
     3121 agaaaaggag ccgatcttct tgaaggactt tctccgatcg tcctcgtact tctcggtcac
     3181 cacggccttc ttcttcaggt ggatatcgtc atactcggca ttccacttgt cccggatcac
     3241 gttccactcg ccgaagatat ccttggagat tgtgctgatg gcggggccgt tcttcacaaa
     3301 gatgccggcg ctagagtact cgtcaaaatt cttgaacagc ttctccagct tcttgatgga
     3361 gctgaagatc tcgctgttct tgttcagggt gtttctaaac acctccagca cctcctcatc
     3421 ggatgtatag ccctcgccgt agaagctcag agactcccga tcgctcagca cctgcttata
     3481 cagtggctta aacttaggca gcttctgctt ggttttctga ttatacaggt tgatgtactc
     3541 gttcaggccc ttgatcttct cgccgctctc ggtcacgaag ccgccgatga tggcgttata
     3601 cacgtcgatg ccctcctgtg tcagcacaaa gttaaagaac tcgccctcaa agaaatcctc
     3661 cacatcatag tcgctgttca ggatcttctc cttgatctcc tgcacctcgt gcttatcaaa
     3721 gatggcgtcc accttctcga agatgtccat attagagatg tagcgggtca gattctcgtt
     3781 gatacacctg aaggcgatgg atgtgctctt ggcctcctcg gaaaacatat tctctctgtt
     3841 cctaaagaag ccggtgaagg ctgtggtaaa gccattgaag ctgttcacca gggcgatctc
     3901 gtccttatcg tccaggaact ctggcaggat tgtctcgatg atatccttct taaacaggga
     3961 cttgtagccc tcgttgccct tgaaggcctt ggcgatctcc ttccgcagat tgatctccag
     4021 gttctccagc tccttattct ccttctcggt tctggttttc ttccggaaca ggctgatgta
     4081 attgttcaga ttcttcagct tgatgctgtg cagcacgtcg ttgataaaag acagatagta
     4141 gcgatccagc agcttcttca cgcccttata atcctcggct ctcttctcgt cctccaccag
     4201 cagccgctta ttgtcgatgt tctcctgggt cttgcccaca gggatggcct tgaacctcag
     4261 ggtcttagac agggagtagc agtttgtaaa cttctccagc ttgctcatgt tattatctag
     4321 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc tctagagccg tccagctcga
     4381 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg aattcgaagt
     4441 tcctattctc tagaaagtat aggaacttcc cggccagatc actagtgatc cagacatgat
     4501 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4561 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4621 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     4681 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatctct agtcaaggca
     4741 ctatacatca aatatttttc cataattttc ttgtatagca gtgcagcttt ttcctttgtg
     4801 gtgtaaatag caaagcaagc aagagttcta ttactaaaca cagcatgact caaaaaactt
     4861 agcaattctg aaggaaagtc cttggggtct tctacctttc tcttcttttt tggaggagta
     4921 gaatgttgag agtcagcagt agcctcatca tcactagatg gcatttcttc tgagcaaaac
     4981 aggttttcct cattaaaggc attccaccac tgctcccatt catcagttcc ataggttgga
     5041 atctaaaata cacaaacaat tagaatcagt agtttaacac attatacact taaaaatttt
     5101 atatttacct tagagcttta aatctctgta ggtagtttgt ccaattatgt cacaccacag
     5161 aagtaaggtt ccttcacaaa gatccttatt cctgtttacg cttctcgggc cgcggcttgt
     5221 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     5281 gatcgcgctt ctcgttgggg tctttgctca gcttggactg ggtgctcagg tagtggttgt
     5341 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
     5401 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct
     5461 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
     5521 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     5581 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     5641 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     5701 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     5761 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     5821 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     5881 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc attttggaag
     5941 ttcctattct ctagaaagta taggaacttc gtttggtatg cgtccccaat tccctattca
     6001 gagttctctt cttgtattca ataattactt cttggcagat ttcagtagtt gcagttgatt
     6061 tacttggttg ctggttactt ttaattgatt cactttaact tgcactttac tgcagattgt
     6121 ttagcttgtt cagctgcgct tgtttatttg cttagctttc gcttagcgac gtgttcactt
     6181 tgcttgtttg aattgaattg tcgctccgta gacgaagcgc ctctatttat actccggcgc
     6241 tcgctagagt ctccgctcgg aggacagtac tccgctcgga ggacagtact ccgctcggag
     6301 gacagtactc cgctcggagg acagtactcc gctcggagga cagtactccg acctgcaggc
     6361 atgcaagctt ggatccgcta gcataacttc gtatagcata cattatacga agttatgcta
     6421 gcctagttcc agtgaaatcc aagcattttc taaattaaat gtattcttat tattatagtt
     6481 gttatttttg atatatataa acaacactat tatgcccacc atttttttga gatgcatcta
     6541 cacaaggaac aaacactgga tgtcactttc agttcaaatt gtaacgctaa tcactccgaa
     6601 caggtcacaa aaaattacct taaaaagtca taatattaaa ttagaataaa tatagctgtg
     6661 agggaaatat atacaaatat attggagcaa ataaattgta catacaaata tttattacta
     6721 atttctattg agacgaaatg aaccactcgg aaccatttga gcgaaccgaa tcgcgcggaa
     6781 ctaacgacag tcgctccaag gtcgtcgaac aaaaggtgaa tgtgttgcgg agagcgggtg
     6841 ggagacagcg aaagagcaac tacgaaacgt ggtgtggtgg aggtgaatta tgaagagggc
     6901 gcgcgatttg aaaagtatgt atataaaaaa tatatcccgg tgttttatgt agcgataaac
     6961 gagtttttga tgtaaggtat gcaggtgtgt aagtcttttg gttagaagac aaatccaaag
     7021 tctacttgtg gggatgttcg aaggggaaat acttgtattc tataggtcat atcttgtttt
     7081 tattggcaca aatataatta cattagcttt ttgagggggc aataaacagt aaacacgatg
     7141 gtaataatgg taaaaaaaaa aaacaagcag ttatttcgga tatatgtcgg ctactccttg
     7201 cgtcgggccc gaagtcttag agccagatat gcgagcaccc ggaagctcac gatgagaatg
     7261 gccagaccca cgtagtccag cggcagatcg gcggcggaga agttaagcgt ctccaggatg
     7321 accttgcccg aactggggca cgtggtgttc gacgatgtgc agctaatttc gcccggctcc
     7381 acgtccgccc attggttaat cagcagaccc tcgttggcgt aacggaacca tgagaggtac
     7441 gacaaccatt tgaggtatac tggcaccgag cccgagttca agaagaagcc gccaaagagc
     7501 aggaatggta tgataaccgg cggacccaca gacagcgcca tcgaggtcga ggagctggcg
     7561 caggatatta gatatccgaa ggacgttgac acattggcca ccagagtgac cagcgccagg
     7621 cagttgaaga agtgcagcac tccggcccgc agtccgatca tcggataggc aatcgccgtg
     7681 aagaccagtg gcactgtgag aaaaagcggc aattcggcaa tcgttttgcc cagaaagtat
     7741 gtgtcacagc gataaagtcg acttcgggcc tccctcataa aaactggcag ctctgaggtg
     7801 aacacctaaa tcgaatcgat tcattagaaa gttagtaaat tattgaaatg caaatgtatt
     7861 ctaaacatga cttacattta tcgtggcaaa gacgttttga aaggtcatgt tggtcaggaa
     7921 gaggaagatg gctccgttga tattcatcac acccacttgc gtgagttgtt ggcccaaaaa
     7981 gatgaggcca atcaagatgg caaccatctg caaattaaaa tgttactcgc atctcattaa
     8041 tattcgcgag ttaaatgaaa tttatttatc ttctgcaaaa ctataaacta tacatctcat
     8101 tgaaaaaaac taagaagggt gtggaatcag gcaattctat ctaaaatcta gcgaatttgt
     8161 ttccaagaat tgtaagcgtt atatcatttg tttccactgg aaccactcac cgttgtctga
     8221 ataagtcgca cttttacgag gagtggttcc ttgagcaccg acagccagga tcgccacagg
     8281 accgcccgga actgcatgaa ccaggtggcc ttgtaggtgt acccattctc cggctgctcc
     8341 agtggcttct ccagattttt ggtggccaac aactgctcca tatcccgggc tactttgcta
     8401 atggcaaaat tgtcgcatat cttggcgatc cgatcacggg actcgatctc ccgtccgggc
     8461 acaacggcca acacctgtac gtaaaagtcc gccggattgt agttggtagg acactgggca
     8521 cccacgctgg ataggagttg agatgttatg taatactaga tacccttaat aaacacatcg
     8581 aactcactag gaaaagaagt cgacggcttc gctgggagtg cccaagaaag ctaccctgcc
     8641 ctcggccatc agaaggatct tgtcaaagag ctcaaacagc tcggaagacg gctgatgaat
     8701 ggtcaggatg acggtcttgc ccttctgcga cagcttcttc agcacctgga cgacgctgtg
     8761 ggcggtaaag gagtccagtc cggaggtggg ctcatcgcag atcagaagcg gcggatcggt
     8821 tagagcctcg gaggcgaatg ccagacgctt cctttctccg ccggacagac ctttcaccct
     8881 gccgggcaca ccgatgatcg tgtgctgaca tttgctgagc gaaagctcct ggatcacctg
     8941 atccacgcgg gccactcgct gccgataggt cagatgtcgt ggcatccgca ccatggcttg
     9001 gaaaatcagg tgttccctgg ccgttaggga gccgataaag aggtcatcct gctggacata
     9061 ggcgcacctg gcctgcatct ccttggcgtc cacaggttgg ccattgagca gtcgcatccc
     9121 ggatggcgat acttggatgc cctgcggcga tcgaaaggca agggcattca gcagggtcgt
     9181 ctttccggca ccggaactgc ccatcacggc caaaagttcg cccggatagg ccacgccgca
     9241 aactgagttt caaattggta attggaccct ttattaagat ttcacacaga tcagccgact
     9301 gcgaatagaa actcaccgtt cttgagcaaa tgtttcctgg gcgccggtat gtgtcgctcg
     9361 ttgcagaata gtccgcgtgt ccggttgacc agctgccgcc atccggagcc cggctgattg
     9421 accgccccaa agatgtccat attgtgccag gcataggtga ggttctcggc tagttggccg
     9481 ctccctgaac cggagtcctc cggcggactg ggtggccgga gcgtgccgta gtttttggcc
     9541 tgcccgaagc cctggttaat gcagctctgc gaagccgctc cgctgtcacc ctgcaatgat
     9601 aggggatctc aaatatcaac tacaagcgtt atgctcatct aaccccgaac aaaaagtacc
     9661 ccgaagtatc ctacgaagta ggtttatact tttatttatt ttttgtgcat ctaggatcag
     9721 cttaaaatat ctggttgtta tattttttgt aaaaaagaat atagtcgaaa atgaatgcct
     9781 ttagatgtct tgatcatgat atgatctcaa aaattgtctt atatagcgag aacagctacc
     9841 agaataatct gtttcgtgtc actatttgtt tgtgcaattg cggtttggga tttttgtggg
     9901 tcgcagttct cacgccgcag acaatttgat gttgcaatcg cagttcctat agatcaagtg
     9961 aacttaagat gtatgcacat gtactactca cattgttcag atgctcggca gatgggtgtt
    10021 tgctgcctcc gcgaattaat agctcctgat cctcttggcc cattgccggg atttttcaca
    10081 ctttcccctg cttacccacc caaaaccaat caccacccca atcactcaaa aaacaaacaa
    10141 aaataagaag cgagaggagt tttggcacag cactttgtgt ttaattgatg gcgtaaaccg
    10201 cttggagctt cgtcacgaaa ccgctgacaa aatgcaactg aaggcggaca ttgacgctac
    10261 gtaacgctac aaacggtggc gaaagagata gcggacgcag cggcgaaaga gacggcgata
    10321 tttctgtgga cagagaagga ggcaaacagc gctgactttg agtggaatgt cattttgagt
    10381 gagaggtaat cgaaagaacc tggtacatca aatacccttg gatcgaagta aatttaaaac
    10441 tgatcagata agttcaatga tatccagtgc agtaaaaaaa aaaaatgttt tttttatcta
    10501 ctttccgcaa aaatgggttt tattaactta catacatact aggggcccgg tacgtaccca
    10561 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
    10621 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
    10681 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
    10741 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
    10801 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
    10861 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
    10921 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
    10981 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
    11041 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
    11101 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
    11161 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
    11221 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
    11281 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
    11341 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
    11401 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
    11461 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
    11521 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
    11581 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
    11641 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
    11701 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
    11761 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
    11821 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
    11881 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
    11941 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
    12001 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
    12061 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
    12121 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
    12181 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
    12241 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
    12301 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
    12361 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
    12421 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
    12481 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
    12541 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
    12601 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
    12661 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
    12721 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
    12781 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
    12841 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
    12901 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
    12961 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
    13021 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
    13081 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
    13141 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
    13201 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
    13261 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
    13321 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctcgaaat
    13381 taaccctcac taaagggaac aaaagctggc tagaactagt gtcgacatgc ccgccgtgac
    13441 cgtcgagaac ccgctgacgc tgccccgcgt atccgcaccc gccgacgccg tcgcacgtcc
    13501 cgtgctcacc gtgaccaccg cgcccagcgg tttcgagggc gagggcttcc cggtgcgccg
    13561 cgcgttcgcc gggatcaact accgccacct cgacccgttc atcatgatgg accagatggg
    13621 tgaggtggag tacgcgcccg gggagcccaa gggcacgccc tggcacccgc accgcggctt
    13681 cgagaccgtg acctacatcg tcgacactag tggatccaga catgataaga tacattgatg
    13741 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
    13801 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
    13861 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
    13921 acctct
//
