LOCUS       sequence-491683-       10337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human codon optimized engineered goldDn29 
            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-goldDn29-dCas9-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..10337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      638..1226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1127..1146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(1359..1434)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1455..1703)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1513..1532)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1683..1703)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1754..1771
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1816..2994
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2047..2985
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2942..2962
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3001..3010
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4567..4587
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4714..4734
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4744..4767,4768..4770,4771..7257,7258..7260,
                     7261..8844)
                     /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             4744..8844
                     /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             8848..8868
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8884..8937
                     /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(8938..8940,8941..9654)
                     /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(8938..8940,8941..9651)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9241..9260)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9588..9609
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    9686..9807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9723..9742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9777..9796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        9839..9943
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9944..10012,10013..10337,1..467)
                     /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(10162..10181)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
       61 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
      121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
      181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
      241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
      301 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
      361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
      421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
      481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
      541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
      601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
      661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      781 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     1261 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     1321 accgtattac cgcctttgcg catcagaatt caaaaaaagc accgactcgg tgccactttt
     1381 tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta aaacacaaca
     1441 gcagaataca cactggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     1501 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     1561 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     1621 acagtcaaat taattccaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     1681 ggaatcatgg gaaataggcc ctcgctagct gaagtgagct gataccgctc gccgcagccg
     1741 aacgaccgag cgcagcgagt cagtgagcga ggaagggaag agcggacctt ctaggtcttg
     1801 aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     1861 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     1921 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     1981 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2041 acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct
     2101 tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg
     2161 gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct
     2221 tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc
     2281 gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg
     2341 acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt
     2401 tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg
     2461 aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg
     2521 cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg
     2581 gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg
     2641 agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg
     2701 aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg
     2761 tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag
     2821 gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct
     2881 tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt
     2941 ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgacgtacg
     3001 gccaccatgg aacgggtgct catacacctt agaaagtcca gagccgatct ggaagctgag
     3061 gccaggggcg agggcgaaac cctggctaaa catagaaaca tcctgctgaa gctggccaaa
     3121 gaaagaaatc tgaacatcgt gaagatcaga gaggaagtgg gcatcagcgg cgagtctctg
     3181 atccacagac ctgagatgct ggagacactg aagggggtgg aagagggact gtacgacgct
     3241 gtgctgtgca tggacatcga cagactgggc agaggcaata ccaaggagca gggcattatc
     3301 ctggaggcct tcaagaatag caacaccaag atcatcaccc ctctgaaaac atacgacctg
     3361 aacaacgagt tcgacgagga atacgccgag tttggcgcct tcatggccag gcgggaattc
     3421 aagtttatca ccagaagact gcagcggggc agagtggcca cagttgagga gggaaattac
     3481 atcgctccaa gaccacctta cggctacatc atcgagaaaa acaacaagga gcggtacctg
     3541 attcctcacc ctgagcaggc ccctatcgtg aagatgatct ttgactggta cacccacgac
     3601 gatcctaacg tgcggatggg cgccagcaag atcgccaacg agctgaacaa gttctgcaag
     3661 agccctacag gcatcccctg gaaggtctcc accgtgttgt ctatcttgaa gaatgccgtg
     3721 tacgccggca gaatccagtg gcggaagaag gaggaaaaaa aatctatcac accaggcaaa
     3781 aaaaaggatg tccggatgcg gcccaaggaa caatggatcg acgtggaggg caagcacgaa
     3841 cctctggtgt ccatggaaac ctacctgaag gcccaagaga tcctggagcg gaagtaccac
     3901 gtgccctatc agatccagaa cggcatcact aaccccctgg ccggactggt caagtgcggg
     3961 atttgtggta gcagcatggt gtatagacct tacacccaca agcgccaccc ccacctgatc
     4021 tgctacaaaa gatactgtac caacaagtct agcagattcg agtacgtgga aagcaaaatt
     4081 atccaaggcc tgcaccagct gctggcacag tacaaggccg attggttcaa acatcagaga
     4141 cccaaggtga acgacgacag cgtggacctg agacagaaag ccccccaccg gctggagaag
     4201 gaactgaatg acctgtacaa gcagaaggac aacctgcaca atctgctgga gaagggcgtg
     4261 tacagcatcg atacattcct ggaaagaagc aacatcctgg ctgaacgcat cgatgatacc
     4321 aagaaggcca tccacgacgc cgagaaggct ctggccgagg aggtgcagcg gaacaaagtg
     4381 aagaaggata tcatccccac agtcgagaac gtgcttgaac tgtactacaa gacccaggac
     4441 cctgccaaga agaacaacct gctgaagagc gtgctggact acgctgttta cagaaaagaa
     4501 aagcaccaga gaaacgatga cttcaccttg gtgctttatc ctaagctgcc tcagatcaac
     4561 agcggaccta agaaaaagag gaaggtgtct ggtagcgaga cacccggtac cagtgaaagc
     4621 gccacgccag aaagcagtgg gagtgagact ccgggtacat ctgaatcagc gacaccggaa
     4681 tcaagtggcg gcagcagcgg cggcagcagc agccccaaga agaagagaaa ggtggaggcc
     4741 agcgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
     4801 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     4861 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     4921 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     4981 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     5041 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     5101 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     5161 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     5221 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     5281 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     5341 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     5401 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac
     5461 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     5521 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     5581 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     5641 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     5701 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     5761 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     5821 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     5881 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     5941 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     6001 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     6061 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     6121 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     6181 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     6241 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     6301 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     6361 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     6421 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     6481 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     6541 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     6601 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     6661 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     6721 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     6781 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     6841 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     6901 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     6961 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     7021 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     7081 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     7141 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     7201 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgct
     7261 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     7321 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     7381 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     7441 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     7501 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     7561 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     7621 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     7681 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     7741 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     7801 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     7861 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     7921 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     7981 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     8041 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     8101 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     8161 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     8221 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     8281 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     8341 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     8401 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     8461 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     8521 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     8581 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     8641 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     8701 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     8761 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     8821 gacctgtctc agctgggagg cgacggacct aagaaaaaga ggaaggtggc tagcggcagt
     8881 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccagtg
     8941 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     9001 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     9061 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     9121 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     9181 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     9241 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     9301 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     9361 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     9421 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     9481 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     9541 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     9601 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagaattc
     9661 gatatcaagc ttatcgataa tcaacaactt gtttattgca gcttataatg gttacaaata
     9721 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     9781 tttgtccaaa ctcatcaatg tatcttagac gtcaggtggc acttttcggg gaaatgtgcg
     9841 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     9901 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     9961 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
    10021 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
    10081 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
    10141 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
    10201 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
    10261 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
    10321 catgagtgat aacactg
//
