LOCUS       sequence-344992-       10219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor shuttle plasmid to enable ligation of a second U6-GuideRNA 
            casssette into any pX458 family backbone via digestion of both 
            plasmids with XbaI and KpnI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10219)
  TITLE     Brzezinski Dual Guide Donor/Activator/Repressor plasmids
REFERENCE   2  (bases 1 to 10219)
  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"
COMMENT     Sequence Label: pX458-Dual-Guide-Donor-Cas9-H2B-mCherry
FEATURES             Location/Qualifiers
     source          1..10219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     513..532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        680..928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     680..700
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     851..870
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        947..1022
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1270..2452
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1501..2443
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2400..2420
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2458..2467
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2467..2532
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2539..2559
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2584..6684
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2584..6684
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6685..6732
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6748..6801
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6802..7179
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      7192..7201
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7198..7905
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7343..7361)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7933..7950)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7939..8146
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8155..8295
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8155..8284
                     /label=AAV2 ITR
     rep_origin      8370..8825
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8457..8476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8667..8688
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8842..8861)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8961..8983
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9021..9039)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9107..9211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9212..9280,9281..10072)
                     /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(9430..9449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcatc tagacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      721 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      781 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      841 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      901 atatatcttg tggaaaggac gaaacaccgg gtcttcgaga agacctgttt tagagctaga
      961 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1021 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
     1081 gtgcgccaat tctgcagaca aatggctcta caggtaccag taattcatac aaaaggactc
     1141 gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt agaacccaga
     1201 gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg tggagaagag
     1261 catgcgtgag gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     1321 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     1381 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     1441 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     1501 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     1561 ccttgaatta cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt
     1621 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
     1681 agttgaggcc tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     1741 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     1801 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
     1861 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     1921 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
     1981 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     2041 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     2101 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     2161 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     2221 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     2281 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     2341 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     2401 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaccggtgcc
     2461 accatggact ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac
     2521 gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
     2581 gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     2641 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     2701 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     2761 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     2821 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     2881 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     2941 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     3001 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     3061 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     3121 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     3181 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     3241 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     3301 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     3361 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     3421 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     3481 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     3541 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     3601 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     3661 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     3721 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     3781 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     3841 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     3901 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     3961 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     4021 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     4081 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     4141 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     4201 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     4261 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     4321 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     4381 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     4441 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     4501 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     4561 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     4621 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     4681 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     4741 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     4801 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     4861 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     4921 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     4981 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     5041 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     5101 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     5161 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     5221 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     5281 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     5341 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     5401 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     5461 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     5521 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     5581 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     5641 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     5701 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     5761 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     5821 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     5881 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     5941 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     6001 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     6061 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     6121 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     6181 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     6241 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     6301 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     6361 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     6421 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     6481 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     6541 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     6601 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     6661 gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca
     6721 aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac
     6781 gtcgaggaga atcctggccc aatgcctgaa ccctctaagt ctgctccagc ccctaaaaag
     6841 ggttctaaga aggctatcac taaggcgcag aagaaggatg gtaagaagcg taagcgcagc
     6901 cgcaaggaga gctattctat ctatgtgtac aaggttctga agcaggtcca ccccgacacc
     6961 ggcatctcat ccaaggccat ggggatcatg aactccttcg tcaacgacat cttcgagcgc
     7021 atcgcgggcg aggcttctcg cctggctcac tacaataagc gctcgaccat cacctccagg
     7081 gagattcaga cggctgtgcg cctgctgctg cctggggagc tggctaagca tgctgtgtcc
     7141 gagggcacta aggcagttac caagtacact agctctaagg atccaccggt cgccaccatg
     7201 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     7261 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     7321 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     7381 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     7441 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     7501 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     7561 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     7621 cagaagaaaa ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     7681 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     7741 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     7801 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     7861 gccgagggcc gccactccac cggcggcatg gacgagctgt acaaggaatt ctaactagag
     7921 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     7981 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     8041 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     8101 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     8161 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     8221 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     8281 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     8341 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     8401 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     8461 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     8521 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     8581 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     8641 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     8701 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     8761 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     8821 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     8881 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     8941 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     9001 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     9061 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     9121 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     9181 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     9241 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     9301 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     9361 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     9421 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     9481 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     9541 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     9601 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     9661 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     9721 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     9781 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     9841 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     9901 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     9961 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
    10021 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
    10081 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
    10141 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
    10201 ccactgagcg tcagacccc
//
