LOCUS       sequence-346774-       10170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting human CYP3A4 proximal enhancer right boundary. sgRNA 
            expressing cells could be FACS sorted by BFP expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10170)
  AUTHORS   Ma H, de Zwaan E, Guo YE, Cejas P, Thiru P, van de Bunt M, Jeppesen 
            JF, Syamala S, Dall'Agnese A, Abraham BJ, Fu D, Garrett-Engele C, 
            Lee TI, Long HW, Griffith LG, Young RA, Jaenisch R
  TITLE     The nuclear receptor THRB facilitates differentiation of human PSCs 
            into more mature hepatocytes.
  JOURNAL   Cell Stem Cell. 2022 Apr 13. pii: S1934-5909(22)00150-3. doi: 
            10.1016/j.stem.2022.03.015.
  PUBMED    35452598
REFERENCE   2  (bases 1 to 10170)
  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 Stem 
            Cell. 2022 Apr 13. pii: S1934-5909(22)00150-3. doi: 
            10.1016/j.stem.2022.03.015."
COMMENT     Sequence Label: 330-BFP-hCYP3A4-enhancer-R-sgRNA
FEATURES             Location/Qualifiers
     source          1..10170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        730..1014
                     /label=chicken beta-actin promoter
     intron          1015..1242
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1254..1263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1263..1328
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1335..1355
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1380..5480
                     /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             1380..5480
                     /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             5481..5528
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5556..5573)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5562..5769
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5838..6141
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6142..6345
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6296..6316
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(6374..6376,6377..6379,6380..7093)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     rep_origin      7648..8103
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7735..7754)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7945..7966
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8120..8139)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8239..8261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8299..8317)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8385..8489
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8490..8558,8559..9350)
                     /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(8708..8727)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9521..10109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10010..10029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gaaactcatg tcccaattaa gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg ggggggggcg cgcgssrggc ggggcggggc ggggcgaggg gcggggcggg
      901 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
      961 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     1021 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     1081 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     1141 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
     1201 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca
     1261 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
     1321 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
     1381 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     1441 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     1501 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     1561 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     1621 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     1681 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     1741 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     1801 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     1861 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     1921 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     1981 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     2041 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     2101 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     2161 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     2221 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     2281 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     2341 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     2401 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     2461 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     2521 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     2581 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     2641 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     2701 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     2761 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     2821 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     2881 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     2941 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     3001 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     3061 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     3121 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     3181 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     3241 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     3301 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     3361 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     3421 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     3481 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     3541 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     3601 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     3661 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     3721 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     3781 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     3841 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
     3901 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
     3961 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     4021 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
     4081 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     4141 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     4201 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     4261 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     4321 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     4381 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     4441 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     4501 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     4561 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     4621 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     4681 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
     4741 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     4801 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     4861 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     4921 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     4981 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     5041 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     5101 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     5161 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     5221 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
     5281 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     5341 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     5401 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     5461 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     5521 agaaaaagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     5581 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     5641 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     5701 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     5761 tgctggggag cggccgctag ttattaatag taatcaatta cggggtcatt agttcatagc
     5821 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     5881 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     5941 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     6001 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     6061 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     6121 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     6181 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     6241 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     6301 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     6361 cagatccgct agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     6421 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgaggggcg agggcgaggg
     6481 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     6541 gccctggccc accctcgtga ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc
     6601 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     6661 gcgcaccatc ttcttcaagg acgacggcac ctacaagacc cgcgccgagg tgaagttcga
     6721 gggcgacacc ctagtgaacc gcatcgagct gaagggcgtc gacttcaagg aggacggcaa
     6781 catcctgggg cacaagctgg agtacaactt caacagccac aacatctata tcatggccgt
     6841 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacgtgg aggacggcag
     6901 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     6961 gcccgacagc cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg
     7021 cgatcacatg gtcctgctgg agttccgcac cgccgccggg atcactctcg gcatggacga
     7081 gctgtacaag taaggatccg ggatgcagaa attgatgatc tattaaacaa taaagatgtc
     7141 cactaaaatg gaagtttttc ctgtcatact ttgttaagaa gggtgagaac agagtaccta
     7201 cattttgaat ggaaggattg gagctacggg ggtgggggtg gggtgggatt agataaatgc
     7261 ctgctcttta ctgaaggctc tttactattg ctttatgata atgtttcata gttggatatc
     7321 ataatttaaa caagcaaaac caaattaagg gccagctcat tcctcccact catgatctat
     7381 agatctatag atctctcgtg ggatcattgt ttttctcttg attcccactt tgtggttcta
     7441 agtactgtgg tttccaaatg tgtcagtttc atagcctgaa gaacgagatc agcagcctct
     7501 gttccacata cacttcattc tcagtattgt tttgccaagt tctaattcca tcagaagctg
     7561 gtcgacctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     7621 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     7681 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     7741 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     7801 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     7861 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     7921 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     7981 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     8041 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     8101 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     8161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     8221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     8281 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     8341 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     8401 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     8461 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     8521 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     8581 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     8641 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     8701 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     8761 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     8821 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     8881 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     8941 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     9001 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     9061 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     9121 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     9181 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     9241 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     9301 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     9361 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     9421 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     9481 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     9541 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     9601 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     9661 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     9721 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     9781 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     9841 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     9901 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     9961 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    10021 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    10081 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
    10141 tggccttttg ctggcctttt gctcacatgt
//
