LOCUS       sequence-491689-       10337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human codon optimized engineered superDn29
            recombinase fused to dCas9 and gRNA expression cassette for 
            targeting attH1 with H1-g3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10337)
  AUTHORS   Fanton A, Bartie LJ, Martins JQ, Tran VQ, Goudy L, Kernick C, 
            Durrant MG, Wei J, Armour-Garb Z, Pawluk A, Konermann S, Marson A, 
            Gilbert LA, Roth TL, Hsu PD
  TITLE     Site-specific DNA insertion into the human genome with engineered 
            recombinases.
  JOURNAL   Nat Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3.
  PUBMED    41199024
REFERENCE   2  (bases 1 to 10337)
  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: "Nat 
            Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3."
COMMENT     Sequence Label: pEf1a-superDn29-dCas9-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..10337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(103..122)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      916..1504
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1405..1424
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(1637..1712)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1733..1981)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1791..1810)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1961..1981)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2032..2049
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2094..3272
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2325..3263
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3220..3240
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3279..3288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4845..4865
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4992..5012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(5022..5045,5046..5048,5049..7535,7536..7538,
                     7539..9122)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5022..9122
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9126..9146
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9162..9215
                     /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             join(9216..9218,9219..9932)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(9216..9218,9219..9929)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9519..9538)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9866..9887
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    9964..10085
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10001..10020)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10055..10074
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        10117..10221
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10222..10290,10291..10337,1..745)
                     /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"
ORIGIN
        1 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
       61 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      121 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      181 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      241 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
      301 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
      361 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
      421 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
      481 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
      541 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
      601 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
      661 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
      721 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
      781 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
      841 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
      901 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
      961 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1021 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1081 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1141 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1201 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1261 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1321 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1381 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1441 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1501 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1561 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgcgca
     1621 tcagaattca aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
     1681 ttattttaac ttgctatttc tagctctaaa acacaacagc agaatacaca ctggtgtttc
     1741 gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca
     1801 tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact
     1861 ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attccaatta
     1921 tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct
     1981 cgctagctga agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2041 gtgagcgagg aagggaagag cggaccttct aggtcttgaa aggagtggga attggctccg
     2101 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2161 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2221 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2281 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg
     2341 gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca
     2401 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
     2461 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc
     2521 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
     2581 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
     2641 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
     2701 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     2761 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     2821 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     2881 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     2941 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     3001 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     3061 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     3121 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     3181 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     3241 aaagtttttt tcttccattt caggtgtcgt gacgtacggc caccatggaa cgggtgctca
     3301 tacaccttag aaagtccaga gccgatctgg aagctgaggc caggggcgag ggcgaaaccc
     3361 tggctaaaca tagaaacatc ctgctgaagc tggccaaaga aagaaatctg aacatcgtga
     3421 agatcagaga ggaagtgggc atcagcggcg agtctctgat ccacagacct gagatgctgg
     3481 agacactgaa gggggtggaa gagggactgt acgacgctgt gctgtgcatg gacatcgaca
     3541 gactgggcag aggcaatacc aaggagcagg gcattatcct ggaggccttc aagaatagca
     3601 acaccaagat catcacccct ctgaaaacat acgacctgaa caacgagttc gacgaggaat
     3661 acgccgagtt tggcgccttc atggccaggc gggaattcaa gtttatcacc agaagactgc
     3721 agcggggcag agtggccaca gttgaggagg gaaattacat cgctccaaga ccaccttacg
     3781 gctacatcat cgagaaaaac aacaaggagc ggtacctgat tcctcaccct gagcaggccc
     3841 ctatcgtgaa gatgatcttt gactggtaca cccacgacga tcctaacgtg cggatgggcg
     3901 ccagcaagat cgccaacgag ctgaacaagt tctgcaagag ccctacaggc atcccctgga
     3961 aggtctccac cgtgttgtct aagttgaaga atgccgtgta cgccggcaga atccagtggc
     4021 ggaagcggga ggaaaaaaaa tctatcacac caggcaaaaa aaaggatgtc cggatgcggc
     4081 ccaaggaaca atggatcgac gtggagggca agcacgaacc tctggtgtcc atggaaacct
     4141 acctgaaggc ccaagagatc ctggagcgga agtaccacgt gccctatcag aagcagaacg
     4201 gcatcactaa ccccctggcc ggactggtca agtgcgggat ttgtggtagc agcatggtgt
     4261 atagacctta cacccacaag cgccaccccc acctgatctg ctaccagaga tactgtacca
     4321 acaagtctag cagattcgag tacgtggaaa gcaaaattat ccaaggcctg caccagctgc
     4381 tggcacagta caaggccgat tggttcaaac atcagagacc caaggtgaac gacgacagcg
     4441 tggaccccag acagaaagcc ctccaccggc tggagaagga actgaatgac ctgtgcaagc
     4501 agaaggacaa cctgcacaat ctgctggaga agggcgtgta cagcatcgat acattcctgg
     4561 aaagaagcaa catcctggct gaacgcatcg atgataccaa gaaggccatc cacgacgccg
     4621 agaaggctct ggccgaggag gtgcagcgga acaaagtgaa gaaggatatc atccccacag
     4681 tcgagaacgt gcttgaactg tactacaaga cccaggaccc tgccaagaag aacaacctgc
     4741 tgaagagcgt gctggactac gctgtttaca gaaaagaaaa gcaccagaga gacgatgact
     4801 tcaccttggt gctttatcct aagctgcctc agatcaacag cggacctaag aaaaagagga
     4861 aggtgtctgg tagcgagaca cccggtacca gtgaaagcgc cacgccagaa agcagtggga
     4921 gtgagactcc gggtacatct gaatcagcga caccggaatc aagtggcggc agcagcggcg
     4981 gcagcagcag ccccaagaag aagagaaagg tggaggccag cgacaagaag tacagcatcg
     5041 gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     5101 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     5161 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     5221 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     5281 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     5341 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     5401 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     5461 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     5521 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     5581 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     5641 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     5701 agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc
     5761 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     5821 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     5881 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     5941 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     6001 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     6061 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     6121 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     6181 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     6241 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     6301 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     6361 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     6421 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     6481 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     6541 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     6601 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     6661 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     6721 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     6781 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     6841 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     6901 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     6961 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     7021 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     7081 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     7141 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     7201 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     7261 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     7321 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     7381 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     7441 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     7501 tggacatcaa ccggctgtcc gactacgatg tggacgctat cgtgcctcag agctttctga
     7561 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     7621 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     7681 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     7741 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     7801 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     7861 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     7921 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     7981 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     8041 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     8101 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     8161 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     8221 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     8281 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     8341 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     8401 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     8461 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     8521 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     8581 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     8641 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     8701 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     8761 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     8821 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     8881 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     8941 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     9001 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     9061 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     9121 acggacctaa gaaaaagagg aaggtggcta gcggcagtgg agagggcaga ggaagtctgc
     9181 taacatgcgg tgacgtcgag gagaatcctg gcccagtgag caagggcgag gagctgttca
     9241 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     9301 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     9361 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     9421 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     9481 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     9541 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     9601 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     9661 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     9721 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     9781 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     9841 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     9901 tcactctcgg catggacgag ctgtacaagt aagaattcga tatcaagctt atcgataatc
     9961 aacaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
    10021 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
    10081 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
    10141 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
    10201 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
    10261 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
    10321 gctgaagatc agttggg
//
