LOCUS       sequence-458340-       12461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inducible expression of Cas12a+ with Gal4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12461)
  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 12461)
  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-uMCas12a+nls2x
FEATURES             Location/Qualifiers
     source          1..12461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2255..2273
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     primer_bind     complement(2306..2325)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2307..2325)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2332..2349)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2332..2348)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2341..2363)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2490..2945
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2577..2596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2787..2808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2971..3075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3076..3144,3145..3936)
                     /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(3294..3313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4107..4695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4596..4615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4849..4866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4983..5004
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5019..5024,5025..5042,5043..5049)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5057..5073
                     /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     5062..5084
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5081..5097
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5081..5097
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5118..5136
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    5355..5424
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     polyA_signal    5453..5587
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5477..5496)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5531..5550
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5859..5879)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          6009..6074
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(6161..6208)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6236..6316)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(6323..6370)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6371..10054)
                     /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"
     promoter        complement(10200..10438)
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     complement(10232..10253)
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     protein_bind    10457..10551
                     /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(10584..10617)
                     /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(11392..12006,12077..12208,12412..12461,
                     1..266,328..982,1057..1330,1732..1803))
                     /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"
ORIGIN
        1 acagccagga tcgccacagg accgcccgga actgcatgaa ccaggtggcc ttgtaggtgt
       61 acccattctc cggctgctcc agtggcttct ccagattttt ggtggccaac aactgctcca
      121 tatcccgggc tactttgcta atggcaaaat tgtcgcatat cttggcgatc cgatcacggg
      181 actcgatctc ccgtccgggc acaacggcca acacctgtac gtaaaagtcc gccggattgt
      241 agttggtagg acactgggca cccacgctgg ataggagttg agatgttatg taatactaga
      301 tacccttaat aaacacatcg aactcactag gaaaagaagt cgacggcttc gctgggagtg
      361 cccaagaaag ctaccctgcc ctcggccatc agaaggatct tgtcaaagag ctcaaacagc
      421 tcggaagacg gctgatgaat ggtcaggatg acggtcttgc ccttctgcga cagcttcttc
      481 agcacctgga cgacgctgtg ggcggtaaag gagtccagtc cggaggtggg ctcatcgcag
      541 atcagaagcg gcggatcggt tagagcctcg gaggcgaatg ccagacgctt cctttctccg
      601 ccggacagac ctttcaccct gccgggcaca ccgatgatcg tgtgctgaca tttgctgagc
      661 gaaagctcct ggatcacctg atccacgcgg gccactcgct gccgataggt cagatgtcgt
      721 ggcatccgca ccatggcttg gaaaatcagg tgttccctgg ccgttaggga gccgataaag
      781 aggtcatcct gctggacata ggcgcacctg gcctgcatct ccttggcgtc cacaggttgg
      841 ccattgagca gtcgcatccc ggatggcgat acttggatgc cctgcggcga tcgaaaggca
      901 agggcattca gcagggtcgt ctttccggca ccggaactgc ccatcacggc caaaagttcg
      961 cccggatagg ccacgccgca aactgagttt caaattggta attggaccct ttattaagat
     1021 ttcacacaga tcagccgact gcgaatagaa actcaccgtt cttgagcaaa tgtttcctgg
     1081 gcgccggtat gtgtcgctcg ttgcagaata gtccgcgtgt ccggttgacc agctgccgcc
     1141 atccggagcc cggctgattg accgccccaa agatgtccat attgtgccag gcataggtga
     1201 ggttctcggc tagttggccg ctccctgaac cggagtcctc cggcggactg ggtggccgga
     1261 gcgtgccgta gtttttggcc tgcccgaagc cctggttaat gcagctctgc gaagccgctc
     1321 cgctgtcacc ctgcaatgat aggggatctc aaatatcaac tacaagcgtt atgctcatct
     1381 aaccccgaac aaaaagtacc ccgaagtatc ctacgaagta ggtttatact tttatttatt
     1441 ttttgtgcat ctaggatcag cttaaaatat ctggttgtta tattttttgt aaaaaagaat
     1501 atagtcgaaa atgaatgcct ttagatgtct tgatcatgat atgatctcaa aaattgtctt
     1561 atatagcgag aacagctacc agaataatct gtttcgtgtc actatttgtt tgtgcaattg
     1621 cggtttggga tttttgtggg tcgcagttct cacgccgcag acaatttgat gttgcaatcg
     1681 cagttcctat agatcaagtg aacttaagat gtatgcacat gtactactca cattgttcag
     1741 atgctcggca gatgggtgtt tgctgcctcc gcgaattaat agctcctgat cctcttggcc
     1801 cattgccggg atttttcaca ctttcccctg cttacccacc caaaaccaat caccacccca
     1861 atcactcaaa aaacaaacaa aaataagaag cgagaggagt tttggcacag cactttgtgt
     1921 ttaattgatg gcgtaaaccg cttggagctt cgtcacgaaa ccgctgacaa aatgcaactg
     1981 aaggcggaca ttgacgctac gtaacgctac aaacggtggc gaaagagata gcggacgcag
     2041 cggcgaaaga gacggcgata tttctgtgga cagagaagga ggcaaacagc gctgactttg
     2101 agtggaatgt cattttgagt gagaggtaat cgaaagaacc tggtacatca aatacccttg
     2161 gatcgaagta aatttaaaac tgatcagata agttcaatga tatccagtgc agtaaaaaaa
     2221 aaaaatgttt tttttatcta ctttccgcaa aaatgggttt tattaactta catacatact
     2281 aggggcccgg tacgtaccca attcgcccta tagtgagtcg tattacaatt cactggccgt
     2341 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2401 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2461 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2521 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2581 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2641 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2701 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2761 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2821 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2881 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     2941 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3001 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3061 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3121 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3181 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3241 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3301 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3361 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3421 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3481 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3541 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3601 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3661 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3721 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3781 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3841 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3901 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3961 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4021 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4081 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4141 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4201 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4261 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4321 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4381 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4441 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4501 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4561 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4621 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4681 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4741 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4801 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4861 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4921 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4981 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     5041 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     5101 gattacgcca agctcgaaat taaccctcac taaagggaac aaaagctggc tagaactagt
     5161 gtcgacatgc ccgccgtgac cgtcgagaac ccgctgacgc tgccccgcgt atccgcaccc
     5221 gccgacgccg tcgcacgtcc cgtgctcacc gtgaccaccg cgcccagcgg tttcgagggc
     5281 gagggcttcc cggtgcgccg cgcgttcgcc gggatcaact accgccacct cgacccgttc
     5341 atcatgatgg accagatggg tgaggtggag tacgcgcccg gggagcccaa gggcacgccc
     5401 tggcacccgc accgcggctt cgagaccgtg acctacatcg tcgacactag tggatccaga
     5461 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     5521 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     5581 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     5641 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atctctagtc
     5701 aaggcactat acatcaaata tttttccata attttcttgt atagcagtgc agctttttcc
     5761 tttgtggtgt aaatagcaaa gcaagcaaga gttctattac taaacacagc atgactcaaa
     5821 aaacttagca attctgaagg aaagtccttg gggtcttcta cctttctctt cttttttgga
     5881 ggagtagaat gttgagagtc agcagtagcc tcatcatcac tagatggcat ttcttctgag
     5941 caaaacaggt tttcctcatt aaaggcattc caccactgct cccattcatc agttccatag
     6001 gttggaatct aaaatacaca aacaattaga atcagtagtt taacacatta tacacttaaa
     6061 aattttatat ttaccttaga gctttaaatc tctgtaggta gtttgtccaa ttatgtcaca
     6121 ccacagaagt aaggttcctt cacaaagatc ctctagatta ctttttcttt tttgcctggc
     6181 cggccttttt cgtggccgcc ggcctttttt cctgtttacg cttctcgggc cgcggggcat
     6241 agtcggggac atcatatggg tatgcataat caggcacgtc gtagggataa gcgtaatctg
     6301 gaacatcgta tgggtaggat ccctttttct tttttgcctg gccggccttt ttcgtggccg
     6361 ccggcctttt gtgcttcacg ctggtctggg cgtactccag ccactccttg ttagagatgg
     6421 cgatcttcac cttatccagc ttctcgtcct cggccttctt gaactggccg atggcccaca
     6481 gcacctttct ggcgatgtta taggcgccat tggcgtcggc gttctttggc aggatggcat
     6541 tctcctgggc ctcatagttc cggctatcgt agaagatgcc gtcggagttc ttcacagggc
     6601 tgatcagaaa atccacgtcg gtgcggcctg tgatgctgtt ccgcatctgc agcatcaggc
     6661 tcatcagggc cataaagcta gagtagaagg ccttgtcgga ctgctcgcac agcagggctc
     6721 tgatatcgcc ctgctgataa ttgatgccgt acttgttgaa cagctcctta taggcgctgg
     6781 tcaggcacac ctcctcccag tcgaacacgt tgttcttctt aggattccgg aagattctga
     6841 tccggttgcc gtaggagtac agcttccact tcttgatgta atcggcgtct gtgcgagaga
     6901 agttcttata gtccagggca aactcgaaca gatcctcctc gggcacgtac atgatcctgt
     6961 caaaggagct gatgaacttc ttggaatcgg cgatgctggt atacttggtt ttcagcaggt
     7021 tcacaaagcc ggtagatgga tcgatcttgg atgtcagcca ggcagggatg taaaagatga
     7081 agccgttctg ggtagacatg gacttaaagc tctcgaactt attggtgatc tgatagccct
     7141 tcagggcgcc gcctgttgca caaggattag acttcttgtc caccatgtag ttcagcttat
     7201 cgatcagcat cttctcgaac ttctgataca cctgcttctc caccttcacg cggctattct
     7261 taaagccaga gttcaggtcc tccagggcga tcacggcatc gtacttctcc accagctcgc
     7321 agatcttgtg caccacctga gagatatagc cggccttcag ctccttgata ttctcgatgg
     7381 aggtccagtt ctggcgggcc tcgaacctct ccttctcctt cttgtccagc agagagtggt
     7441 aatctgtctt gatcctgatg ccgttgaagt tgttgatgat ctcgttcagg gaatactgct
     7501 ccacgatgtt gcccttgccg tccaccacca cgatatacag cagattgcgc tcgcccctat
     7561 cgatgccgat cacatagggg ttatcgtcgt gcttcagcag cacgcgcacc tctgtattga
     7621 tcttgaagat gttcttgggg cacttattga tggcgattgg gatgtgcagc tcgtactggt
     7681 cctcagaaaa cctcttatcc ttatacacgt cgtaggacag ggttgtggtt ttcttgggat
     7741 tatctggatt cttgttggcg ataggggagt tggctgggtg caccaccagc tcctccttct
     7801 tcagggaggc gcgcctcatg aacagctctg ctcctccgct cagcctgatc tgtccgtgat
     7861 tgttctcgtc aaacagcagc ttgaagtaca tggtgtgcag attgggtgtg ccgtgagact
     7921 tatcggaaaa gtccttgtta tagatctgga acatatacag cttgccctcc tccaccagct
     7981 tatccacctc cttcttgctg gcagactcga agctcacctt atagccctgc tcctccacct
     8041 ctctgtaaaa gccggcgatg tccttatact tctctgtctc agaaaagttg aaatcgtagg
     8101 cattggacca ctttggatac cgggagatgc tatccttaaa gaagtcgatc agcttgtgac
     8161 agtcattcag gttaaacata tcgcccttct tgaatgtgcc attcttgtag atcttctgga
     8221 tgtcctcgct ggggttatag taggccatcc acttcttaga aaagaacacc tttggcagca
     8281 tcttattagg gccgggcagc agcttatagt tgatcttctc gtaattgccg ttcacatcgt
     8341 ccttgtcgat cttctgcagg cacttggcgt acttcttatc catgatggcc agatagtact
     8401 tggagccgta tctcaggatg gtggcccgat agtctgtctc cttatccttg tcccagccgc
     8461 ccatgaactg agggttctga aaatacagct tgaacttatc cttagagtag ggcttctggg
     8521 tcacataatt gcggatggca tcgtagatgt ggtccacctt cagcaggatg tcgtaggcca
     8581 gcacaaaatc gccatagaag gactcgtccc tgtttgtctc cttgccctcg ccaaagaagg
     8641 ccttgatgta attctcgaag ctcttcacag aatccagcag gtccttcatg atggccacca
     8701 cggcgtcgtt cttcttcagg ctcttctcca gcacaaaatc ggcgtcgaac agcttctcag
     8761 aggagccata caccttgtag atctcatcca ccttctggat gatgatctcc ttcagcttct
     8821 ccaccacaga cagatcggcg tcggcgtact cctgcagctg ctccagagaa aaggagccga
     8881 tcttcttgaa ggactttctc cgatcgtcct cgtacttctc ggtcaccacg gccttcttct
     8941 tcaggtggat atcgtcatac tcggcattcc acttgtcccg gatcacgttc cactcgccga
     9001 agatatcctt ggagattgtg ctgatggcgg ggccgttctt cacaaagatg ccggcgctag
     9061 agtactcgtc aaaattcttg aacagcttct ccagcttctt gatggagctg aagatctcgc
     9121 tgttcttgtt cagggtgttt ctaaacacct ccagcacctc ctcatcggat gtatagccct
     9181 cgccgtagaa gctcagagac tcccgatcgc tcagcacctg cttatacagt ggcttaaact
     9241 taggcagctt ctgcttggtt ttctgattat acaggttgat gtactcgttc aggcccttga
     9301 tcttctcgcc gctctcggtc acgaagccgc cgatgatggc gttatacacg tcgatgccct
     9361 cctgtgtcag cacaaagtta aagaactcgc cctcaaagaa atcctccaca tcatagtcgc
     9421 tgttcaggat cttctccttg atctcctgca cctcgtgctt atcaaagatg gcgtccacct
     9481 tctcgaagat gtccatatta gagatgtagc gggtcagatt ctcgttgata cacctgaagg
     9541 cgatggatgt gctcttggcc tcctcggaaa acatattctc tctgttccta aagaagccgg
     9601 tgaaggctgt ggtaaagcca ttgaagctgt tcaccagggc gatctcgtcc ttatcgtcca
     9661 ggaactctgg caggattgtc tcgatgatat ccttcttaaa cagggacttg tagccctcgt
     9721 tgcccttgaa ggccttggcg atctccttcc gcagattgat ctccaggttc tccagctcct
     9781 tattctcctt ctcggttctg gttttcttcc ggaacaggct gatgtaattg ttcagattct
     9841 tcagcttgat gctgtgcagc acgtcgttga taaaagacag atagtagcga tccagcagct
     9901 tcttcacgcc cttataatcc tcggctctct tctcgtcctc caccagcagc cgcttattgt
     9961 cgatgttctc ctgggtcttg cccacaggga tggccttgaa cctcagggtc ttagacaggg
    10021 agtagcagtt tgtaaacttc tccagcttgc tcatgttatt atctagcgac gtaaacggcc
    10081 acaagttcag cgtgtccggc gagggctcta gagccgtcca gctcgaccag gatgggcacc
    10141 accccggtga acagctcctc gcccttgctc accatggtgg gaattcccaa ttccctattc
    10201 agagttctct tcttgtattc aataattact tcttggcaga tttcagtagt tgcagttgat
    10261 ttacttggtt gctggttact tttaattgat tcactttaac ttgcacttta ctgcagattg
    10321 tttagcttgt tcagctgcgc ttgtttattt gcttagcttt cgcttagcga cgtgttcact
    10381 ttgcttgttt gaattgaatt gtcgctccgt agacgaagcg cctctattta tactccggcg
    10441 ctcgctagag tctccgctcg gaggacagta ctccgctcgg aggacagtac tccgctcgga
    10501 ggacagtact ccgctcggag gacagtactc cgctcggagg acagtactcc gacctgcagg
    10561 catgcaagct tggatccgct agcataactt cgtatagcat acattatacg aagttatgct
    10621 agcctagttc cagtgaaatc caagcatttt ctaaattaaa tgtattctta ttattatagt
    10681 tgttattttt gatatatata aacaacacta ttatgcccac catttttttg agatgcatct
    10741 acacaaggaa caaacactgg atgtcacttt cagttcaaat tgtaacgcta atcactccga
    10801 acaggtcaca aaaaattacc ttaaaaagtc ataatattaa attagaataa atatagctgt
    10861 gagggaaata tatacaaata tattggagca aataaattgt acatacaaat atttattact
    10921 aatttctatt gagacgaaat gaaccactcg gaaccatttg agcgaaccga atcgcgcgga
    10981 actaacgaca gtcgctccaa ggtcgtcgaa caaaaggtga atgtgttgcg gagagcgggt
    11041 gggagacagc gaaagagcaa ctacgaaacg tggtgtggtg gaggtgaatt atgaagaggg
    11101 cgcgcgattt gaaaagtatg tatataaaaa atatatcccg gtgttttatg tagcgataaa
    11161 cgagtttttg atgtaaggta tgcaggtgtg taagtctttt ggttagaaga caaatccaaa
    11221 gtctacttgt ggggatgttc gaaggggaaa tacttgtatt ctataggtca tatcttgttt
    11281 ttattggcac aaatataatt acattagctt tttgaggggg caataaacag taaacacgat
    11341 ggtaataatg gtaaaaaaaa aaaacaagca gttatttcgg atatatgtcg gctactcctt
    11401 gcgtcgggcc cgaagtctta gagccagata tgcgagcacc cggaagctca cgatgagaat
    11461 ggccagaccc acgtagtcca gcggcagatc ggcggcggag aagttaagcg tctccaggat
    11521 gaccttgccc gaactggggc acgtggtgtt cgacgatgtg cagctaattt cgcccggctc
    11581 cacgtccgcc cattggttaa tcagcagacc ctcgttggcg taacggaacc atgagaggta
    11641 cgacaaccat ttgaggtata ctggcaccga gcccgagttc aagaagaagc cgccaaagag
    11701 caggaatggt atgataaccg gcggacccac agacagcgcc atcgaggtcg aggagctggc
    11761 gcaggatatt agatatccga aggacgttga cacattggcc accagagtga ccagcgccag
    11821 gcagttgaag aagtgcagca ctccggcccg cagtccgatc atcggatagg caatcgccgt
    11881 gaagaccagt ggcactgtga gaaaaagcgg caattcggca atcgttttgc ccagaaagta
    11941 tgtgtcacag cgataaagtc gacttcgggc ctccctcata aaaactggca gctctgaggt
    12001 gaacacctaa atcgaatcga ttcattagaa agttagtaaa ttattgaaat gcaaatgtat
    12061 tctaaacatg acttacattt atcgtggcaa agacgttttg aaaggtcatg ttggtcagga
    12121 agaggaagat ggctccgttg atattcatca cacccacttg cgtgagttgt tggcccaaaa
    12181 agatgaggcc aatcaagatg gcaaccatct gcaaattaaa atgttactcg catctcatta
    12241 atattcgcga gttaaatgaa atttatttat cttctgcaaa actataaact atacatctca
    12301 ttgaaaaaaa ctaagaaggg tgtggaatca ggcaattcta tctaaaatct agcgaatttg
    12361 tttccaagaa ttgtaagcgt tatatcattt gtttccactg gaaccactca ccgttgtctg
    12421 aataagtcgc acttttacga ggagtggttc cttgagcacc g
//
