LOCUS       sequence-421525-       11407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA-EFS-Cas9-P2A-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11407)
  AUTHORS   Tan IL, Perez AR, Lew RJ, Sun X, Baldwin A, Zhu YK, Shah MM, Berger 
            MS, Doudna JA, Fellmann C
  TITLE     Targeting the non-coding genome and temozolomide signature enables 
            CRISPR-mediated glioma oncolysis.
  JOURNAL   Cell Rep. 2023 Nov 28;42(11):113339. doi: 
            10.1016/j.celrep.2023.113339. Epub 2023 Nov 2.
  PUBMED    37917583
REFERENCE   2  (bases 1 to 11407)
  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: "Cell Rep.";
            date: "2023-11-28"; volume: "42(11):113339. doi"; pages: " 
            10.1016/j.celrep.2023.113339. Epub 2023 Nov 2"
COMMENT     Sequence Label: pCF827-recipient_U6-sgRNA-EFS-Cas9-P2A-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..11407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        232..611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     766..786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             829..1009
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1056..1181
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1674..1907
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2092..2136
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2434..2551
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2602..2850
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2602..2622
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2773..2792
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3025..3236
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3255..3264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3261..7364
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3261..7364
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7365..7412
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7413..7436
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7446..7502
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     misc_feature    8229..8817
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8282..8302)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8700..8711)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8889..9122
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9148..9165)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9154..9378
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(9484..9501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9655..10243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9735..9754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10414..11205,11206..11274))
                     /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     11037..11056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11275..11379)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat tgacattgat
      241 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      301 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      361 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      421 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      481 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      541 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      601 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      661 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      721 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      781 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      841 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      901 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      961 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     1021 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     1081 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     1141 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1201 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1261 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1321 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1381 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1441 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1501 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1561 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1621 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1681 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1741 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1801 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1861 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1921 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1981 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     2041 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     2101 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     2161 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     2221 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     2281 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     2341 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     2401 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2581 agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata tttgcatata
     2641 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2701 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2761 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2821 ttatatatct tgtggaaagg acgaaacacc ggagacggag gacgacgaac gtctctgttt
     2881 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     2941 aaaagtggca ccgagtcggt gctttttttg aattcgctag ctaggtcttg aaaggagtgg
     3001 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     3061 tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg
     3121 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     3181 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc
     3241 ggttctagag cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca
     3301 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     3361 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     3421 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     3481 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     3541 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     3601 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     3661 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     3721 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     3781 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     3841 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     3901 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     3961 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     4021 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     4081 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     4141 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     4201 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     4261 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     4321 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     4381 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     4441 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     4501 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     4561 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     4621 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     4681 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     4741 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     4801 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     4861 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     4921 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     4981 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     5041 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     5101 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     5161 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     5221 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     5281 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     5341 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     5401 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     5461 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     5521 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     5581 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     5641 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     5701 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     5761 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     5821 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     5881 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     5941 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     6001 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     6061 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     6121 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     6181 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     6241 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     6301 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     6361 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     6421 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     6481 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     6541 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     6601 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     6661 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     6721 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     6781 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     6841 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     6901 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     6961 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     7021 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     7081 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     7141 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     7201 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     7261 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     7321 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca
     7381 caaagaaggc tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat
     7441 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     7501 cgatggtcag taagggcgaa gaagataata tggcttcctt gcctgccaca catgaactcc
     7561 acatattcgg gtctatcaac ggcgtggact tcgacatggt tgggcaagga accggaaatc
     7621 ccaacgatgg atacgaggag ctgaacctga agtcaacgaa aggtgatctt caattctccc
     7681 cctggattct cgtccctcac attggctatg gtttccacca gtacctcccg taccctgacg
     7741 ggatgtctcc attccaagcc gcaatggtag atgggtcagg ctaccaagtt cacagaacta
     7801 tgcagtttga agatggtgca tctctgacag taaattacag gtatacctac gaaggttcac
     7861 acatcaaagg agaagctcag gtgaaaggta cgggattccc agcagacggc ccagtaatga
     7921 ccaattccct cacggctgcc gactggtgca ggagtaaaaa gacatatcca aatgacaaga
     7981 ctattatctc tactttcaaa tggtcttata cgacaggcaa cggaaagagg tatcggtcaa
     8041 ccgcgcgcac aacatatacg tttgctaagc ctatggctgc caattatctg aaaaatcagc
     8101 ccatgtatgt atttagaaaa accgaactga agcactccaa aactgaattg aacttcaaag
     8161 agtggcaaaa ggccttcacc gatgtcatgg gcatggatga attgtataag tgaacgcgtt
     8221 aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     8281 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     8341 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     8401 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     8461 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     8521 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     8581 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     8641 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     8701 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     8761 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc
     8821 gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     8881 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     8941 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     9001 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     9061 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     9121 cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     9181 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     9241 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     9301 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     9361 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ccattaatga aaagaaccag
     9421 ccattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     9481 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     9541 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     9601 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     9661 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     9721 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     9781 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     9841 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     9901 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     9961 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
    10021 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
    10081 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
    10141 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
    10201 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
    10261 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
    10321 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
    10381 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
    10441 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
    10501 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
    10561 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
    10621 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
    10681 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
    10741 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
    10801 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
    10861 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
    10921 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
    10981 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
    11041 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
    11101 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
    11161 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
    11221 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
    11281 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
    11341 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
    11401 acctgac
//
