LOCUS       sequence-421511-       11402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA-EFS-Cas9-P2A-mCherry2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11402)
  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 11402)
  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: pCF826-sgNT-1_U6-sgRNA-EFS-Cas9-P2A-mCherry2
FEATURES             Location/Qualifiers
     source          1..11402
                     /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        3020..3231
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3250..3259
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3256..7359
                     /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             3256..7359
                     /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             7360..7407
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7408..7431
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7441..7497
                     /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    8224..8812
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8277..8297)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8695..8706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8884..9117
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9143..9160)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9149..9373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(9479..9496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9650..10238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9730..9749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10409..11200,11201..11269))
                     /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     11032..11051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11270..11374)
                     /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 gggccaaacg tgccctgacg ggtttaagag
     2881 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2941 tggcaccgag tcggtgcttt ttttgaattc gctagctagg tcttgaaagg agtgggaatt
     3001 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     3061 ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     3121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     3181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggaccggttc
     3241 tagagcgctg ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct
     3301 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     3361 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     3421 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     3481 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     3541 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     3601 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     3661 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     3721 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     3781 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     3841 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     3901 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     3961 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     4021 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     4081 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     4141 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     4201 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     4261 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     4321 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     4381 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     4441 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     4501 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     4561 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     4621 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     4681 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     4741 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     4801 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     4861 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     4921 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     4981 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     5041 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     5101 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     5161 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     5221 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     5281 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     5341 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     5401 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     5461 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     5521 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     5581 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     5641 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     5701 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     5761 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     5821 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     5881 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     5941 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     6001 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     6061 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     6121 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     6181 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     6241 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     6301 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     6361 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     6421 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     6481 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     6541 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     6601 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     6661 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     6721 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     6781 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     6841 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     6901 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     6961 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     7021 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     7081 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     7141 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     7201 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     7261 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     7321 tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc cgccacaaag
     7381 aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa gggatccggc
     7441 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg
     7501 gtcagtaaag gtgaagagga caatatggct atcatcaagg agttcatgcg ctttaaggta
     7561 cacatggaag gctcagttaa cgggcatgag ttcgaaatag agggagaagg tgaaggacgc
     7621 ccctatgaag gcacccaaac ggctaagctg aaggtaacta agggcggtcc cttgcccttc
     7681 gcctgggaca tactgagccc gcagttcatg tacggttcca aagcctatgt aaaacacccc
     7741 gccgacattc ctgactatct taagctgtct tttcccgaag gctttaactg ggaaagagta
     7801 atgaatttcg aggatggagg ggtggtcact gtaacacagg attctagtct gcaagatggg
     7861 gagttcatct acaaggtgaa gctgcgcggc acgaattttc cctcagatgg tcctgtcatg
     7921 cagtgtcgga ctatggggtg ggaagcaagc acagaaagaa tgtatcctga ggacggggca
     7981 ctgaagggag agatcaaaca gcggctcaaa cttaaagacg gcgggcacta tgacgccgaa
     8041 gttaagacaa cttacaaggc caagaagccc gttcagttgc cgggcgcata caacgtggac
     8101 ataaagctcg acatcttgtc ccataatgag gactacacaa tcgtggaaca gtatgaacgg
     8161 gccgagggca gacattctac cggggggatg gatgagcttt acaagtaaac gcgttaagtc
     8221 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     8281 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     8341 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     8401 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     8461 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     8521 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     8581 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8641 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8701 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8761 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     8821 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     8881 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     8941 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     9001 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     9061 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
     9121 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     9181 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     9241 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     9301 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     9361 gtgggctcta tggcttctga ggcggaaaga accagccatt aatgaaaaga accagccatt
     9421 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     9481 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     9541 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     9601 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     9661 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     9721 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     9781 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     9841 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     9901 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     9961 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
    10021 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
    10081 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
    10141 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
    10201 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
    10261 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
    10321 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
    10381 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
    10441 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
    10501 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
    10561 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
    10621 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
    10681 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
    10741 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
    10801 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
    10861 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
    10921 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
    10981 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
    11041 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
    11101 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
    11161 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
    11221 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
    11281 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
    11341 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
    11401 ac
//
