LOCUS       sequence-484543-       20281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning in complete guide expression cassettes, with Cas12a (D156R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 20281)
  AUTHORS   Lawrenson T, Clarke M, Kirby R, Forner M, Steuernagel B, Harwood W
  TITLE     An Optimised CRISPR Cas9 and Cas12a Toolkit for Barley and Wheat
  JOURNAL   Research Square, 12 March 2024, PREPRINT
REFERENCE   2  (bases 1 to 20281)
  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: "Research 
            Square, 12 March 2024, PREPRINT"
COMMENT     Sequence Label: EC67841
FEATURES             Location/Qualifiers
     source          1..20281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    32..56
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             365..994
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             1423..2496
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGASSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      2562..2756
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     promoter        2818..2922
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2923..3717
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3888..4476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4377..4396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4612..4636
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    4724..4898
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     intron          5620..5809
                     /label=cat1 intron
                     /note="castor bean catalase intron, modified"
     terminator      complement(8037..8289)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(8305..8352)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    15083..15104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(15119..15124,15125..15142,15143..15149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15157..15173
                     /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)."
     RBS             15191..15202
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             15209..15886
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      15931..16002
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      16018..16045
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      19995..20247
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 cacgaagtga tccgtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa
       61 gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg
      121 ttcgtccatt tgtatgtgca tgccaaccac agggttcccc tcgggagtca gccgtgcggc
      181 tgcatgaaat cctggccggt ttgtctgatg ccaagctggc ggcctggccg gccagcttgg
      241 ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag taaaacagct
      301 tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata cgcaagggga
      361 acgcatgaag gttatcgctg tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc
      421 aacccatcta gcccgcgccc tgcaactcgc cggggccgat gttctgttag tcgattccga
      481 tccccagggc agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt
      541 cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt
      601 agtgatcgac ggagcgcccc aggcggcgga cttggctgtg tccgcgatca aggcagccga
      661 cttcgtgctg attccggtgc agccaagccc ttacgacata tgggccaccg ccgacctggt
      721 ggagctggtt aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt
      781 gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta
      841 cgagctgccc attcttgagt cccgtatcac gcagcgcgtg agctacccag gcactgccgc
      901 cgccggcaca accgttcttg aatcagaacc cgagggcgac gctgcccgcg aggtccaggc
      961 gctggccgct gaaattaaat caaaactcat ttgagttaat gaggtaaaga gaaaatgagc
     1021 aaaagcacaa acacgctaag tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg
     1081 ttggccagcc tggcagacac gccagccatg aagcgggtca actttcagtt gccggcggag
     1141 gatcacacca agctgaagat gtacgcggta cgccaaggca agaccattac cgagctgcta
     1201 tctgaataca tcgcgcagct accagagtaa atgagcaaat gaataaatga gtagatgaat
     1261 tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga
     1321 atgccccatg tgtggaggaa cgggcggttg gccaggcgta agcggctggg ttgtctgccg
     1381 gccctgcaat ggcactggaa cccccaagcc cgaggaatcg gcgtgagcgg tcgcaaacca
     1441 tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc
     1501 gcgcaggccg cccagcggca acgcatcgag gcagaagcac gccccggtga atcgtggcaa
     1561 gcggccgctg atcgaatccg caaagaatcc cggcaaccgc cggcagccgg tgcgtcgtcg
     1621 attaggaagc cgcccaaggg cgacgagcaa ccagattttt tcgttccgat gctctatgac
     1681 gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt
     1741 gaccgacgag ctggcgaggt gatccgctac gagcttccag acgggcacgt agaggtttcc
     1801 gcagggccgg ccggcatggc cagtgtgtgg gattacgacc tggtactgat ggcggtttcc
     1861 catctaaccg aatccatgaa ccgataccgg gaagggaagg gagacaagcc cggccgcgtg
     1921 ttccgtccac acgttgcgga cgtactcaag ttctgccggc gagccgatgg cggaaagcag
     1981 aaagacgacc tggtagaaac ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt
     2041 acgaagaagg ccaagaacgg ccgcctggtg acggtatccg agggtgaagc cttgattagc
     2101 cgctacaaga tcgtaaagag cgaaaccggg cggccggagt acatcgagat cgagctagct
     2161 gattggatgt accgcgagat tacagaaggc aagaacccgg acgtgctgac ggttcacccc
     2221 gattactttt tgatcgatcc cggcatcggc cgttttctct accgcctggc acgccgcgcc
     2281 gcaggcaagg cagaagccag atggttgttc aagacgatct acgaacgcag tggcagcgcc
     2341 ggagagttca agaagttctg tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg
     2401 gagtacgatt tgaaggagga ggcggggcag gctggcccga tcctagtcat gcgctaccgc
     2461 aacctgatcg agggcgaagc atccgccggt tcctaatgta cggagcagat gctagggcaa
     2521 attgccctag caggggaaaa aggtcgaaaa agcttctttc ctgtggatag cacgtacatt
     2581 gggaacccaa agccgtacat tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac
     2641 attgggaacc ggtcacacat gtaagtgact gatataaaag agaaaaaagg cgatttttcc
     2701 gcctaaaact ctttaaaact tattaaaact cttaaaaccc gcctggcctg tgcataactg
     2761 tctggccagc gcacagccga acagctgcaa aaagcgccca catcaaggct ccgagtgcgc
     2821 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2881 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatggctaa aatgagaata
     2941 tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac ggaaggaatg
     3001 tctcctgcta aggtatataa gctggtggga gaaaatgaaa acctatattt aaaaatgacg
     3061 gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat gatgctatgg
     3121 ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg aacggcatga tggctggagc
     3181 aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga agatgaacaa
     3241 agccctgaaa agattatcga gctgtatgcg gagtgcatca ggctctttca ctccatcgac
     3301 atatcggatt gtccctatac gaatagctta gacagccgct tagccgaatt ggattactta
     3361 ctgaataacg atctggccga tgtggattgc gaaaactggg aagaggacac tccatttaaa
     3421 gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga acttgtcttt
     3481 tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt aagtggcttt
     3541 attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt ctgcgtccgg
     3601 tcgctcaggg aggatatcgg ggaagaacag tatgtcgagc tattttttga cttactgggg
     3661 atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt gttttagctg
     3721 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3781 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4021 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4501 cggttcctgc tcggatctgt tggaccggac agtagtcatg gttgatgggc tgcctgtatc
     4561 gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat
     4621 atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt
     4681 aatgtactgg ggttgaacac tctgtgccga attcggatcc agcgatctgg attttagtac
     4741 tggattttgg ttttaggaat tagaaatttt attgatagaa gtattttaca aatacaaata
     4801 catactaagg gtttcttata tgctcaacac atgagcgaaa ccctatagga accctaattc
     4861 ccttatctgg gaactactca cacattatta tggagaaact cgagcttgtc gatcgactct
     4921 agctagagaa gcctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc
     4981 ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt
     5041 acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc
     5101 tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag
     5161 catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg
     5221 cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta
     5281 ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc
     5341 cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc
     5401 ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc
     5461 catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca
     5521 tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag
     5581 atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagttatc atcatcatca
     5641 tagacacacg aaataaagta atcagattat cagttaaagc tatgtaatat ttacaccata
     5701 accaatcaat taaaaaatag atcagtttaa agaaagatca aagctcaaaa aaataaaaag
     5761 agaaaagggt cctaaccaag aaaatgaagg agaaaaacta gaaatttacc tgcagaacag
     5821 cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc
     5881 aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg
     5941 ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta
     6001 cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct
     6061 ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag
     6121 acgtcgcggt gagttcaggc tttttcattc tgcaaaagag aaccagacaa cagggtaagt
     6181 gcctagcagt aaacaaacag aactcatcac aagcaaacag caacatcata ttcataccaa
     6241 caggtcatgt gtgttcatca catcattagt actaagcatg ccatcatcca agtatatcaa
     6301 agtaagggca aagagcatgc atgatcatca ggtgcacaaa agaatcatca aattgtagca
     6361 gtacatatct tcatctatca tgcatatcta tccataacag gacgatgcat gttgaccagg
     6421 taaaagctac aggatcctat aggaacagca gcgtatatat cttcaccaat cttagcatct
     6481 taatcatgtg gcacatgcag tttcaattta agcacatgag ctagttgatt atgaggtacc
     6541 agagaatcat caaactgatg tagcagcata tatcctcatc tatcatgcaa tctaatctaa
     6601 tctaactaaa caggaaaggt gtgctattca gttaaaagct accgcatcat atacaaacgg
     6661 cagcataaga aaaagcataa tcatcttaat catgcaacaa acgcagattc ataataagcc
     6721 caagagctag cttgtgatga tcttattcta ctctgatcta cagcaatcag ataacgacct
     6781 aaccttgcac atggcaacaa aacaatcgat cggacgaatc agttgtttgt tcctagctag
     6841 caccatcgaa ccagataata gatgcacgta cagatcccga aaacgaaccc aaaaacaggg
     6901 cagacctagc tgaacctagg cagcgaccca gcagatcgtg agaacgatct catctacgaa
     6961 cagcctagaa gcaaccccac gattcccgga caaacgacct aaaatcccca caaatcacat
     7021 gagcatgaca ggataaacag cggaaccgat cagatctaca cgaaaacccc acctcccagc
     7081 cacccacgat caggaaacac gcggatctag catgatttcg tcaacgcctc agcctagttc
     7141 ctagccacag accaagcaga accaccaaac cacgccgagc gaggagatgg ggcaagagga
     7201 cgggggacac gatcgccgta ccttgaagcg ggggaaggat cgccgagggt cgcgagaaga
     7261 gcaatttgga tttggaaccc gggggttgtg cgctccgaac gatcagacga tgtgagattg
     7321 tgggaagagg cgcggagggc cctgtattta tgggctgcga cggggggagg agaggtgggg
     7381 agggttgggg aaggaatccc ccacccgtgc cgtgacggtt ccgggccgtg tgagaggagc
     7441 ccgctcgtgt ccgccacgca atttccgcga tcggagcgga gctttcgaga ggcggctgga
     7501 tggttggtgg ccgttagatt tgtagacgcc gttaacgcct cgcctccacc gggaagagtt
     7561 ttgagcagcc gcttatgaca atggcttaac gacgttagac ggagcgttag tggcaggcca
     7621 tgatagaata gacgtattgc aatgggatta taattaaata aataagaata taataagata
     7681 tggcaagtcg gcactcatga catggtgtcc gaaatgatag tgctcacttt cttagccgag
     7741 aaagttgacg cgactgattt agaagttaag atattatttc tctcttcttt tctttcctcg
     7801 tcatataagg atgaaataaa ctttagagat tgccggtagt gattttggat ttcggcgatc
     7861 aggcttggtt tgccggtttc ggacggtgtg ccttaggcca cccgcagtgt atcttgtaat
     7921 gttcaaccga taagcaaggg tggggctcaa gcaagtagta aacaactatg tcaaatgtca
     7981 ccatggttat ggtcttgttt agttggcttc ctccgcaaga attcaagctt agcgtcgatc
     8041 tagtaacata gatgacaccg cgcgcgataa tttatcctag tttgcgcgct atattttgtt
     8101 ttctatcgcg tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa
     8161 taacgtcatg cattacatgt taattattac atgcttaacg taattcaaca gaaattatat
     8221 gataatcatc gcaagaccgg caacaggatt caatcttaag aaactttatt gccaaatgtt
     8281 tgaacgatct gcttgacaag ctcatttctt cttcttagcc tgaccagctt tcttagtagc
     8341 agcaggcctc ttgtgcttaa cggaggtctg agcgtactca agccactctt tgttggagat
     8401 ggcgatcttc accttgtcca gcttctcatc ttccgctttc ttaaactgcc cgatagccca
     8461 gagaacctat aacacgggag aaaccaaaaa gcaaactcat ttcacgccaa atactacaga
     8521 tttctcaaaa attaagttat aatccatata tcctgacaag aaattgatta aaacacagtt
     8581 gctttacctt tctcgcaatg ttgtaagccc cgtttgcatc agcattcttc ggcaggatag
     8641 cgttctcttg agcctcatag ttacggctgt cgtagaagat cccgtcagag ttcttgaccg
     8701 gagagataag gaagtccaca tcggtccttc cggtgataga gttcctcatc tggagcatga
     8761 gagacatgag ggccatgaat gagctgtaga atgccttgtc gctctgctcg cacaaaagag
     8821 ccctgatatc accctgctgg tagttgatgc cgtacttgtt gaacagttct ttgtaggcag
     8881 aggtgaggca cacttcttcc cagtcgaaca cgttgttttt cttcggattt cggaagatcc
     8941 tgatacggtt cccgtaggag tagagcttcc acttcttaat gtagtcggcg tcggtcctgc
     9001 tgaagttctt atagtcgaga gcgaactcga aaagatcctc ttccggcacg tacatgatcc
     9061 tgtcgaaaga cgagatgaac ttcttgctgt cggcgatcga ggtgtacttg gtcttgagca
     9121 ggttcacgaa accagtagaa ggatcgatct tgctggtgag ccaagcaggg atatagaaga
     9181 tgaagccgtt ctgggtgctc atgctcttga aggactcgaa cttgttcgtg atctggtatc
     9241 ccttaagagc accaccggta gcgcaagggt tgctcttttt gtccaccatg tagttgagct
     9301 tgtcgatgag catcttctcg aacttttggt agacctgctt ctcaacctgt agcaataatt
     9361 aaaacaaaaa taccacaaac agttagaact gaggcaagaa gaatgaatgc agagaaaaaa
     9421 aatcttagct attcatggaa atggaagtgc gacgaacacc taaaatgcaa ggagcataac
     9481 caaatgcaga acttaccttc actctactgt tcttgaaccc agagttgagg tcctcaagag
     9541 cgatcacagc atcgtacttt tccaccagct cgcagatctt atgcaccacc tgagagatgt
     9601 agccggcctt caactctttg atgttttcga tagaagtcca gttctgcctg gcctcgaaac
     9661 gctctttttc tttcttatcg aggagtgagt ggtagtcggt cttgatcctg atgccgttga
     9721 aattgttgat aatctcgttc aggctgtact gctcgacgat gtttcccttt ccgtccacca
     9781 caacgatgta gaggaggttt ctctcaccac gatcgattcc gatcacgtaa gggttatcat
     9841 cgtgcttgag cagcaccctc acctttaatt gtagaaaacc agtaaaacat ggacaattag
     9901 gaggcatcga ttatctaacc aaagacttta acaacactaa tctgccacta atattcatga
     9961 gaagtcctta cctcggtatt gatcttgaag atgttcttcg ggcacttgtt gatagcgatc
    10021 gggatatgaa gctcgtactg gtcctcgctg aaccttttgt ccttgtagac gtcgtaagac
    10081 agcgtggtgg tctttttcgg gttgtcagga ttcttgttag cgattggaga gttggccggg
    10141 tgaacaacca actcttcttt cttgagtgag gccctacgca tgaagagttc agcaccacca
    10201 gaaagtctga tctgcccgtg gttgttctcg tcgaaaagca gcttgaagta catcgtatgg
    10261 aggttaggag ttccgtggct cttgtcggag aagtccttgt tatagatctg gaacatgtag
    10321 agcttgccct cttcgaccaa cttgtcaacc tgttataaca gtaaaaatta ctttagaaac
    10381 aaaacaaaag tgtggtcttt taggaatgat tattgatcag ttcatagttt aaaatcttgt
    10441 gtttttatcg taaaagtgtg gaccaggatt tacctctttc ttggaggcgc tctcgaagct
    10501 aactttgtaa ccctgttcct caacctcacg gtagaacccg gcgatatcct tgtacttttc
    10561 tgtctcgctg aagttaaagt cgtaagcgtt agaccacttc gggtaacgcg agatgctgtc
    10621 cttgaagaaa tcgatgagct tgtggcagtc gttgaggttg aacatgtcgc ctttcttgaa
    10681 ggtcccgttc ttgtagattt tctggatgtc ctcagacggg ttgtagtagg ccatccattt
    10741 cttgctgaag aacaccttag gaagcatctt gttcggtccc ggaaggagct tgtagttgat
    10801 cttctcatag ttcccgttca cgtcatcctt gtcgatcttt tggaggcact tggcgtattt
    10861 cttgtccatg atggccaggt agtacttaga cccgtaccta atgaattaat agaaaaatca
    10921 ggcaaattag aaaagaaata attctcaaaa gagaacaact gaggtcagaa aatagcattc
    10981 atcaactgaa actaggtatt tcttcatatg atactaacct gaggatggtg gccctgtaat
    11041 ctgtctcttt gtctttgtcc catccgccca tgaactgcgg gttctgaaag tacaacttga
    11101 acttgtcctt gctgtaaggc ttctgggtca cgtagtttct gatggcgtcg tagatatggt
    11161 ccaccttgag gaggatatcg taagcgagca cgaaatcccc gtagaaagac tcgtccctat
    11221 tagtctcttt gccctctccg aagaaggcct tgatatagtt ctcgaaggac ttcacgctgt
    11281 cgagcaaatc cttcatgatc gcgacgacag cgtcgttctt cttcagactc ttctcgagga
    11341 cgaaatcagc atcgaacaac ttctcagagg acccgtacac ctaataatat catcagagga
    11401 aaactcaatt cttatatctt acagaaatga acaacaaaat acatgtagca gagaggaaaa
    11461 caatcataag taacaactta ccttgtagat ctcgtcgacc ttctggatga tgatctcttt
    11521 gagcttctcg accacgctga gatcagcatc agcgtattct tgaagctgct cgaggctgaa
    11581 agagccgatc ttcttgaacg acttacgcct gtcatcctcg tacttctcag tgacgacagc
    11641 cttcttcttg aggtggatgt catcgtactc agcattccac ttgtccctga tcacgttcca
    11701 ctcgccgaaa atatccttgc tgatggtgct gatagcaggt ccgttcttaa cgaagatccc
    11761 agcgctgctg tactcgtcga agttcttgaa aagcttctcc aacttcttga tggacgagaa
    11821 gatctcgctg ttcttgttga gggtgttacg gaaaacctca agcacttcct cgtcagaggt
    11881 gtaaccctca ccgtaaaatg agaggctctc acgatcagag agaacctgca agaaacagca
    11941 aagagagagt taagatataa gtgaacaaga aagtcttggg atcgacatac atacaatcaa
    12001 ttgatcaaat tcctaggatt aaacatatga aataggaaag acagacctgc ttgtagagcg
    12061 gcttgaactt cgggagcttc tgcttcgttt tctggttgta caggttgatg tactcgttga
    12121 gtcccttaat cttctcccca gactcggtca cgaatcctcc gataatagcg ttgtacacgt
    12181 cgatgccctc ttgggtgaga acgaagttga agaactcccc ttcaaaaaag tcctcgacgt
    12241 cgtagtcgct gttgaggatc ttctctttga tttcttgcac ctcgtgctta tcgaagatgg
    12301 cgtccacctg agattgtttg caaagaaaaa tctcaaagca tgtacaatct ataaacttat
    12361 gatggaaaaa gaaaagaaag gtaactaagg aatgttacct tctcgaagat gtccatgttg
    12421 ctgatgtaac gcgtgaggtt ctcgttgatg catctgaaag cgatagaggt gctcttggcc
    12481 tcttcgctga acatgttttc cctgtttctg aagaatccgg tgaaggcagt agtgaatccg
    12541 ttgaagctgt tcacgagagc gatctcatcc ttatcgtcca ggaactcagg caggattgtc
    12601 tcgataatat ccttcttgaa gaggctcttg tatccctcgt ttcccttgaa agccttggcg
    12661 atctctttac ggaggttgat ctcgaggttc tcaagctctt tgttctcttt ctcggtcctg
    12721 gttttcttac ggaagaggct gatgtagttg ttgaggttct tgagcttgat gctgtggagc
    12781 acatcgttga tgaagctgag gtagtacctg tcgaggagct tcttcacgcc cttgtaatct
    12841 tcagctctct tctcatcctc gacgaggagc ctcttgttgt cgatattctc ttgggtcttt
    12901 cccacaggga tagccttgaa cctgagggtc ttagagaggc tgtagcagtt ggtaaacttc
    12961 tcgagcttgc tcattcctgc agaagtaaca ccaaacaaca gggtgagcat cgacaaaaga
    13021 aacagtacca agcaaataaa tagcgtatga aggcagggct aaaaaaatcc acatatagct
    13081 gctgcatatg ccatcatcca agtatatcaa gatcaaaata attataaaac atacttgttt
    13141 attataatag ataggtactc aaggttagag catatgaata gatgctgcat atgccatcat
    13201 gtatatgcat cagtaaaacc cacatcaaca tgtataccta tcctagatcg atatttccat
    13261 ccatcttaaa ctcgtaacta tgaagatgta tgacacacac atacagttcc aaaattaata
    13321 aatacaccag gtagtttgaa acagtattct actccgatct agaacgaatg aacgaccgcc
    13381 caaccacacc acatcatcac aaccaagcga acaaaaagca tctctgtata tgcatcagta
    13441 aaacccgcat caacatgtat acctatccta gatcgatatt tccatccatc atcttcaatt
    13501 cgtaactatg aatatgtatg gcacacacat acagatccaa aattaataaa tccaccaggt
    13561 agtttgaaac agaattctac tccgatctag aacgaccgcc caaccagacc acatcatcac
    13621 aaccaagaca aaaaaaagca tgaaaagatg acccgacaaa caagtgcacg gcatatattg
    13681 aaataaagga aaagggcaaa ccaaacccta tgcaacgaaa caaaaaaaat catgaaatcg
    13741 atcccgtctg cggaacggct agagccatcc caggattccc caaagagaaa cactggcaag
    13801 ttagcaatca gaacgtgtct gacgtacagg tcgcatccgt gtacgaacgc tagcagcacg
    13861 gatctaacac aaacacggat ctaacacaaa catgaacaga agtagaacta ccgggcccta
    13921 accatggacc ggaacgccga tctagagaag gtagagaggg gggggggggg aggacgagcg
    13981 gcgtaccttg aagcggaggt gccgacgggt ggatttgggg gagatctggt tgtgtgtgtg
    14041 tgcgctccga acaacacgag gttggggaaa gagggtgtgg agggggtgtc tatttattac
    14101 ggcgggcgag gaagggaaag cgaaggagcg gtgggaaagg aatcccccgt agctgccgtg
    14161 ccgtgagagg aggaggaggc cgcctgccgt gccggctcac gtctgccgct ccgccacgca
    14221 atttctggat gccgacagcg gagcaagtcc aacggtggag cggaactctc gagaggggtc
    14281 cagaggcagc gacagagatg ccgtgccgtc tgcttcgctt ggcccgacgc gacgctgctg
    14341 gttcgctggt tggtgtccgt tagactcgtc gacggcgttt aacaggctgg cattatctac
    14401 tcgaaacaag aaaaatgttt ccttagtttt tttaatttct taaagggtat ttgtttaatt
    14461 tttagtcact ttattttatt ctattttata tctaaattat taaataaaaa aactaaaata
    14521 gagttttagt tttcttaatt tagaggctaa aatagaataa aatagatgta ctaaaaaaat
    14581 tagtctataa aaaccattaa ccctaaaccc taaatggatg tactaataaa atggatgaag
    14641 tattatatag gtgaagctat ttgcaaaaaa aaaggagaac acatgcacac taaaaagata
    14701 aaactgtaga gtcctgttgt caaaatactc aattgtcctt tagaccatgt ctaactgttc
    14761 atttatatga ttctctaaaa cactgatatt attgtagtac tatagattat attattcgta
    14821 gagtaaagtt taaatatatg tataaagata gataaactgc acttcaaaca agtgtgacaa
    14881 aaaaaatatg tggtaatttt ttataactta gacatgcaat gctcattatc tctagagagg
    14941 ggcacgaccg ggtcacgctg cacctccact atgcctgaga cccaatacgc aaaccgcctc
    15001 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
    15061 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
    15121 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
    15181 catactagag aaagaggaga aatactagat ggcttcctcc gaggacgtta tcaaagagtt
    15241 catgcgtttc aaagttcgta tggaaggttc cgttaacggt cacgagttcg aaatcgaagg
    15301 tgaaggtgaa ggtcgtccgt acgaaggtac ccagaccgct aaactgaaag ttaccaaagg
    15361 tggtccgctg ccgttcgctt gggacatcct gtccccgcag ttccagtacg gttccaaagc
    15421 ttacgttaaa cacccggctg acatcccgga ctacctgaaa ctgtccttcc cggaaggttt
    15481 caaatgggaa cgtgttatga acttcgagga cggtggtgtt gttaccgtta cccaggactc
    15541 ctccctgcaa gacggtgagt tcatctacaa agttaaactg cgtggtacca acttcccgtc
    15601 cgacggtccg gttatgcaga aaaaaaccat gggttgggaa gcttccaccg aacgtatgta
    15661 cccggaggac ggtgctctga aaggtgaaat caaaatgcgt ctgaaactga aagacggtgg
    15721 tcactacgac gctgaagtta aaaccaccta catggctaaa aaaccggttc agctgccggg
    15781 tgcttacaaa accgacatca aactggacat cacctcccac aacgaggact acaccatcgt
    15841 tgaacagtac gaacgtgctg aaggtcgtca ctccaccggt gcttaataac gctgatagtg
    15901 ctagtgtaga tcgctactag agccaggcat caaataaaac gaaaggctca gtcgaaagac
    15961 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc
    16021 tcaccttcgg gtgggccttt ctgcgtttat aggtctcagg gattacgaat tcccatgggg
    16081 aggtgcagcg tgacccggtc gtgcccctct ctagagataa tgagcattgc atgtctaagt
    16141 tataaaaaat taccacatat tttttttgtc acacttgttt gaagtgcagt ttatctatct
    16201 ttatacatat atttaaactt tactctacga ataatataat ctatagtact acaataatat
    16261 cagtgtttta gagaatcata taaatgaaca gttagacatg gtctaaagga caattgagta
    16321 ttttgacaac aggactctac agttttatct ttttagtgtg catgtgttct cctttttttt
    16381 tgcaaatagc ttcacctata taatacttca tccattttat tagtacatcc atttagggtt
    16441 tagggttaat ggtttttata gactaatttt tttagtacat ctattttatt ctattttagc
    16501 ctctaaatta agaaaactaa aactctattt tagttttttt atttaataat ttagatataa
    16561 aatagaataa aataaagtga ctaaaaatta aacaaatacc ctttaagaaa ttaaaaaaac
    16621 taaggaaaca tttttcttgt ttcgagtaga taatgccagc ctgttaaacg ccgtcgacga
    16681 gtctaacgga caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg aagcagacgg
    16741 cacggcatct ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca ccgttggact
    16801 tgctccgctg tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag ccggcacggc
    16861 aggcggcctc ctcctcctct cacggcacgg cagctacggg ggattccttt cccaccgctc
    16921 cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc
    16981 ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg
    17041 tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct
    17101 ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt tcatgtttgt
    17161 gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt
    17221 acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg
    17281 gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg
    17341 tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct
    17401 tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga
    17461 tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg
    17521 tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga
    17581 taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct
    17641 tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat
    17701 actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat
    17761 cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg
    17821 atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta
    17881 accttgagta cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat
    17941 atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata
    18001 cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta
    18061 cttctgcagg aatggcgatg ccgtatgcct ctctttcccc ggcaggcgac cgccgctcct
    18121 ccccggccgc caccgccacc gcctccctcc tccccttctg ccgctcctcc cccttctccg
    18181 ccggcggcaa tggcggcatg ggggaggagg cgcggatgga cgggaggtgg atggcgaggc
    18241 cggtgccctt cacggcggcg cagtacgagg agctggagca ccaggcgctc atatacaagt
    18301 acctggtggc cggcgtgtcc gtcccgccgg atctcgtgct ccccatccgc cgcggcatcg
    18361 agtccctcgc cgcccgcttc taccacaacc ccctcgccat cgggtacgga tcgtacctgg
    18421 gcaagaaggt ggatccggag ccgggccggt gccggcgcac ggacggcaag aagtggcggt
    18481 gcgccaagga ggccgcctcc gactccaagt attgcgagcg ccacatgcac cgcggccgca
    18541 accgttcaag aaagcctgtg gaaacgcagc tcgtctcgca ctcccagccg ccggccgcct
    18601 ccgtcgtgcc gcccctcgcc accggcttcc acaaccactc cctctacccc gccatcggcg
    18661 gcaccaacgg tggtggaggc ggggggaaca acggcatgtc catgcccggc acgttctcct
    18721 ccgcgctggg gccgcctcag cagcacatgg gcaacaatgc cgcctctccc tacgcggctc
    18781 tcggcggcgc cggaacatgc aaagatttca ggtataccgc atatggaata agatctttgg
    18841 cagacgagca gagtcagctc atgacagaag ccatgaacac ctccgtggag aacccatggc
    18901 gcctgccgcc atcttctcaa acgactacat tcccgctctc aagctactct cctcagcttg
    18961 gagcaacgag tgacctgggt cagaacaaca gcagcaacaa caacagcggc gtcaaggccg
    19021 agggacagca gcagcagcag ccgctctcct tcccggggtg cggcgacttc ggcagcggcg
    19081 actccgcgaa gcaggagaac cagacgctgc ggccgttctt cgacgagtgg cccaagacga
    19141 gggactcgtg gtcggacctg accgacgaca actcgaacgt cgcctccttc tcggccaccc
    19201 agctgtcgat ctcgataccc atgacgtcct ccgacttctc cgccgccagc tcccagtcgc
    19261 ccaacggcat gctgttcgcc ggcgagatgt acgcggccgc tgccatgcag cagcaacacc
    19321 tgatgcagat gaaccagagc atgatggggg gctacgcttc ctctaccact gccaccactg
    19381 atctcattca gcagtacctg gatgagaaca agcagctgat cctggccatc ctcgacaacc
    19441 agaacaacgg caaggtggag gagtgcgcac ggaaccaagc taagctccag cagaacctca
    19501 tgtacctcgc cgccatcgcc gacagccagc ctccgcagac ggcatcgctg tctcagtacc
    19561 cgtccaacct gatgatgcag tccgggccgc ggtacatgca gcagcagtcg gcgcagatga
    19621 tgtcgccgca gtcgctgatg gcggcgcggt cgtcgatgat gtacgcgcag caggccatgt
    19681 cgccgctcca gcagcagcag cagcagcagc agcaccaggc ggccgcgcac ggccagctgg
    19741 ggatgtcctc cggcgcgacc accgggttca acctcctgca cggtgaggcc agcatgggcg
    19801 gcggcggcgg cgccactggc aacagcatga tgaacgccag cgtcttttcg gactatggcc
    19861 gcggcggcag cggcgccaag gaggggtcga cctcgctgtc ggccgacgct cgcggcgcca
    19921 actctggcgc gcacagcggc gacggggagt acctcaaggg caccgaggag gaaggaagct
    19981 aggcttgtca agcagatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
    20041 ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
    20101 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
    20161 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
    20221 gcgcggtgtc atctatgtta ctagatcgac gctcagagag gatgcacatg tgaccgaggg
    20281 a
//
